Inkt cell modulators and methods of using the same

ABSTRACT

Disclosed herein are α-galactosylceramide (α-GalCer) analogs and compositions thereof, methods of activating invariant Natural Killer T (iNKT) cells using said analogs, methods of treating diseases by activating iNKT cells using said analogs, and combination therapy of said analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/616,672, filed Jun. 7, 2017, now U.S. Pat. No. 10,314,796, which is acontinuation of U.S. patent application Ser. No. 15/153,425, filed May12, 2016, now U.S. Pat. No. 9,700,532, which is a continuation of U.S.patent application Ser. No. 14/361,434, filed May 29, 2014 (§ 371 date),now U.S. Pat. No. 9,365,496, which is the U.S. national stage ofInternational Patent Application No. PCT/EP2012/074140, filed Nov. 30,2012, which claims the benefit of U.S. Provisional Application No.61/565,287, filed Nov. 30, 2011, the disclosure of which is incorporatedby reference in its entirety.

BACKGROUND

Natural killer T (NKT) cells have been implicated in a range ofimportant immune surveillance mechanisms, such as host defense againstexternal pathogens, immune tolerance and malignancy. NKT cells can befurther divided into two subsets, so-named Type I and Type II. Type INKT cells have received the most attention. These cells are also knownas invariant NKT (iNKT) cells owing to their expression of an invarianta chain T cell receptor (TCR; Vα14-Jα18 chain in mice and Vα24-Jα18chain in humans), which is paired with a more variable β chain. Incontrast, Type II NKT cells have a diverse TCR repertoire and are lesswell defined, although a subset has been shown to be reactive tosulfatide. The iNKT cell TCR recognizes lipid antigens presented in thecontext of the non-polymorphic MHC class I-like protein, CD1d. The CD1dmolecule has been shown to bind a range of dialkyl lipids andglycolipids and the ensuing iNKT cell TCR recognition of the CD1d-lipidcomplex leads to the rapid proliferation and release of a plethora ofcytokines (both pro-inflammatory and regulatory). The activation of iNKTcells is an important step in ‘boosting’ adaptive immune responsesthrough the activation and maturation of dendritic cells (DC) and Bcells through CD40-CD40L interactions, and the activation of naturalkiller (NK) cells following interferon gamma (IFNγ) release. Since thestructure of CD1d ligands has been shown to govern the released cytokineprofile, the development of lipid molecules that promote the specificactivation of iNKT cells, could find very useful application in thetreatment of a wide range of disorders.

Of the range of lipids that bind to CD1d, the glycolipidα-galactosylceramide (α-GalCer) is one of the most potent. α-GalCer is aderivative of the agelasphins, which are naturally occurring glycolipidsthat were isolated from the marine sponge Agelas mauritianus.Recognition of the α-GalCer-CD1d complex by the iNKT cell TCR results inthe secretion of a range of cytokines, and the initiation of a powerfulimmune response.

While α-GalCer remains one of the most potent iNKT cell agonists and hasshown potential in the treatment of various conditions, it may provedifficult to use this molecule widely as a useful therapeutic agent, atleast as a direct activator of iNKT cells: not only doesα-GalCer-mediated iNKT cell activation lead to the secretion of both Thelper Type 1 (Th1) (e.g. IFN-γ) and T helper Type 2 (Th2) (e.g.interleukin-4 (IL-4)) cytokines, and therefore a mixed immune response,but more importantly over-stimulation of iNKT cells, which can result intheir entering a long-term anergic state, i.e. unresponsiveness tosubsequent α-GalCer stimulation and preferential IL-4 production, whichwould be deleterious for long-term therapy. Loss of circulating levelsof iNKT cells could represent a therapeutically significant limitationwith iNKT-cell-based therapies if multi-dosing regimens are required.Thus, a need exists for other iNKT cell activators.

SUMMARY

This invention provides compounds, compositions of matter, and methodsof making and using the compounds and compositions that are useful inrelation to iNKT activiation and all of the applications (includingtherapeutic or prophylactic medical applications) relating to iNKTactivation.

By way of example, compounds are disclosed having a structure of formula(I):

wherein R¹ is C₅-C₂₅ alkyl, C₅-C₂₅ alkenyl, C₅-C₂₅ alkynyl, C₅-C₂₅heteroalkyl, C₅-C₂₅ heteroalkenyl, or C₅-C₂₅ heteroalkynyl; R² and R³are each independently selected from H, OH, SH, amino or substitutedamino; R⁴ is C₅-C₂₀ alkyl, C₅-C₂₀ alkenyl, C₅-C₂₀ alkynyl, C₅-C₂₀heteroalkyl, C₅-C₂₀ heteroalkenyl, or C₅-C₂₀ heteroalkynyl; R⁶ and R⁵are each independently selected from H, alkyl, and alkenyl, or R⁶ and R⁵together form a 6-, 7-, or 8-membered cycloalkyl or cycloalkenyl ring; Xis O, S, SO₂, SO(NH), SO(N(alkyl)), NH, N(alkyl), or CH₂; Y is O, NH,N(alkyl), or S; Z is O, S, NH, N(alkyl), or CH₂; with the proviso thatwith the proviso that (a) when Y and X are each O and R⁵ and R⁶ are eachH, Z is not CH₂; and (b) when Y is O, R⁶ and R⁵ together form a6-membered cycloalkyl ring, and Z is CH₂, the cycloalkyl ring is notsubstituted with —CH₂OH, —OH, —CH₃, or —CH₂OCH₃, or a salt, ester,solvate, or hydrate thereof. In some embodiments, the compoundsdisclosed herein having a structure (IA), (IB), (IC), or (ID):

wherein n is 1, 2, or 3; m is 0, 1, or 2; p is 1 or 2; and the dashedline is an optional double bond. In some specific sets of embodiments,the dashed line is a double bond, while in other sets of embodiments,the dashed line is a single bond.

In various cases, the compounds disclosed herein have stereochemistry asnoted in the structure (IE):

In various cases, the compound has a structure of:

wherein n is 1, 2, or 3, or a salt, ester, solvate, or hydrate thereof,or more specifically a structure of:

wherein n is 1 (IMM60), 2 (IMM70), or 3 (IMM80), or a salt, ester,solvate, or hydrate thereof.

In some variations, the compound is purified and/or isolated.

Further disclosed herein are compositions that comprise a compound asdisclosed herein and one or more pharmaceutically acceptable diluents,excipients, or carriers (pharmaceutical compositions). In somevariations, the composition is formulated and/or packaged as a unit dosefor administration to a subject. In some variations, a syringe or otheradministration device is provided that contains the compound orcomposition.

Also disclosed herein are methods of activating an NKT cell bycontacting the cell with a compound or composition as disclosed herein.The activating of the NKT cell can comprise one or more of inducingsecretion of a cytokine from the NKT cell, stimulating proliferation ofthe NKT cell, and upregulating expression of a cell surface marker onthe NKT cell. The cytokine can be one or more of IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, IL-13, IL-15, TNF-α, TNF-β, and IFN-γ. The activating cancomprise upregulating at least one cell surface marker selected fromCD69, CD25, an IL-12 receptor and CD40L.

In some variations, the activating is performed ex vivo, e.g., using abiological sample that contains an NKT cell that has been removed froman organism. In other variations, the activating is performed in vivo,e.g., by administering the compound or composition to the organism by aroute through which the compound or composition or metabolite thereofcontacts the NKT cell.

The methods disclosed herein can comprise administering a compound orcomposition as disclosed herein to a subject in need of NKT cellactivation. Exemplary subjects are mammalian subjects, which includeshuman subjects. In some cases, the subject suffers from cancer.

The methods disclosed herein can comprise administering a compound orcomposition as disclosed herein to a subject suffering from a cancerselected from basal cell carcinoma, breast cancer, leukemia, Burkitt'sLymphoma, colon cancer, esophageal cancer, bladder cancer, gastriccancer, head and neck cancer, hepatocellular cancer, Hodgkin's Lymphoma,hairy cell leukemia, Wilms' Tumor, thyroid cancer, thymoma, thymiccarcinoma, testicular cancer, T-cell lymphoma, prostate cancer,non-small cell lung cancer, liver cancer, renal cell cancer, andmelanoma.

The methods disclosed herein can further comprise administering a secondtherapeutic agent to the subject. For example, the methods can compriseadministering a chemotherapeutic or an immunotherapeutic agent, a cancervaccine, a tumor antigen, or a polynucleotide encoding a tumor antigen.The second therapeutic can be administered simultaneously with thecompound or composition as disclosed herein, and in some specific cases,the two are co-formulated. The second therapeutic can be administeredsequentially with the compound or composition as disclosed herein, e.g.,before or after the compound or composition. Repeated administration ofone or both of the agents is contemplated.

Methods disclosed herein also can be characterized as methods oftreatment or prophylaxis. For example, disclosed herein are methods oftreatment or prophylaxis of a subject suffering from any of theconditions described herein, such method comprising administering to thesubject a compound or composition described herein. In some variations,the compound or composition is administered in an amount effective tostimulate NKT activation. In some variations, other therapeuticbenchmarks are utilized. For example, the compound or composition isadministered in an amount effective to slow the growth, or reduce thesize, or eliminate a tumor or other cancer. In some variations, theadministering is repeated multiple times.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. With respect toaspects of the invention described or claimed with “a” or “an,” itshould be understood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. With respect toelements described as one or more within a set, it should be understoodthat all combinations within the set are contemplated. If aspects of theinvention are described as “comprising” a feature, embodiments also arecontemplated “consisting of” or “consisting essentially of” the feature.

Aspects of the invention described as methods of treatment should alsobe understood to include first or subsequent “medical use” aspects ofthe invention or “Swiss use” of compositions for the manufacture of amedicament for treatment of the same disease or condition.

Multiple embodiments are contemplated for combination inventionsdescribed herein. For example, some aspects of the invention that aredescribed as a method of treatment (or medical use) combining two ormore compounds or agents, whether administered separately (sequentiallyor simultaneously) or in combination (co-formulated or mixed). For eachaspect described in this manner, the invention further includes acomposition comprising the two or more compounds or agents co-formulatedor in admixture with each other; and the invention further includes akit or unit dose containing the two or more compounds/agents packagedtogether, but not in admixture. Optionally, such compositions, kits ordoses further include one or more carriers in admixture with one or bothagents or co-packaged for formulation prior to administration to asubject. The reverse also is true: some aspects of the invention aredescribed herein as compositions useful for therapy and containing twoor more therapeutic agents. Equivalent methods and uses are specificallycontemplated.

Although the applicant(s) invented the full scope of the claims appendedhereto, the claims appended hereto are not intended to encompass withintheir scope the prior art work of others. Therefore, in the event thatstatutory or judicially-recognized prior art within the scope of a claimis brought to the attention of the applicants by a Patent Office orother entity or individual, the applicant(s) reserve the right toexercise amendment rights under applicable patent laws to redefine thesubject matter of such a claim to specifically exclude such prior art orobvious variations of statutory prior art from the scope of such aclaim. Variations of the invention defined by such amended claims alsoare intended as aspects of the invention. Additional features andvariations of the invention will be apparent to those skilled in the artfrom the entirety of this application, and all such features areintended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B the biological activity of six compounds as disclosedherein, α-GalCer, and ThrCer as determined by their ability to stimulateiNKT cell hybridoma DN32, following pulsing of C1R-mCD1d cells with eachcompound, as measured by the resulting IL-2 released in the supernatant.

FIGS. 2A and 2B show the biological activity of the same compoundsasFIGS. 1A and 1B, but this time in a human cell model, with human iNKTcells co-cultured with C1R-hCD1d cells and pulsed with 100 ng/mLcompound or vehicle, and the resulting IL-2 released in the supernatantmeasured.

FIGS. 3A and 3B show results of an experiment of 1 μg lipid injectedinto wildtype C57 BL/6 or C57 BL/6 CD1d−/− (NKT cell-deficient) mice,and IL-4 and IFNγ levels measured.

FIGS. 4A and 4B show fluorescence-activated cell sorting (FACS) analysisof cells harvested from the spleens of the mice in the experiment ofFIGS. 3A-3B, to determine the extent of dendritic cell (DC) maturation.

FIGS. 5A-5B show SPR experiments used to measure the affinity andkinetics of human iNKT cell T cell receptors (TCRs) for hCD1d cellsloaded with α-GalCer, ThrCer, α-GalCer thioamide, α-GalCer carbamate,ThrCer thioamide, and ThrCer carbamate analogues.

FIG. 6. Binding affinity of iNKT TCR to ThrCer6 and ThrCer7 CD1dcomplex. C1R-hCD1d cells were pulsed with various ligands, as noted, andthe affinity of iNKT TCR-tetramer determined by flow cytometry asmeasured by median Fluorescent Intensity (MFI)).

FIG. 7. Binding affinity of iNKT TCR in the presence of differentligands. C1R-hCD1d cells pulsed with various ligands, as noted, and IFNγlevels measured in their supernatant by ELISA.

FIG. 8. Recovery of iNKT cells from activation-induced anergy.Splenocytes cultured for 60 h in the presence of various ligands andIFNγ levels measured in the supernatant by ELISA.

FIGS. 9A and 9B. Adjuvant activity of various ligands. Splenocytes werecontacted for 18 hours with 800 μg OVA, 1 μg lipid, and OVA-specific MHCI (FIG. 9A) and MHCII (FIG. 9B) peptides. IFNγ levels measured after 18hours by ELISpot and expressed as spots per million splenocytes.

FIG. 10. Adjuvant activity of various ligands. Splenocytes werecontacted for 18 hours with 800 μg OVA, 1 μg lipid, and OVA-specific MHCI and MHCII peptides. OVA IgG levels measured after 18 hours.

FIG. 11 contains graphs showing T cell (left panel) and B cell (rightpanel) responses to antigen (OVA) following exposure to various lipids.The data indicate that IMM60 induced stronger T- and B-cell responsescompared to IMM47 and IMM70.

FIG. 12 is a schematic showing the experimental design for theexperiments described below (Section heading: In vivo anergy of iNKTcells).

FIG. 13 is a graph showing the level of IFNγ expression in naïve miceand in mice 18 hours after being immunized with alpha-GalCer, IMM47,IMM60 or IMM70.

FIG. 14 is a graph showing the level of PD1 expression in naïve mice andin mice 28 days after being immunized with alpha-GalCer, IMM47, IMM60 orIMM70.

FIG. 15 is a graph showing the measurement of iNKT cell anergy (asdefined by measurements of IFNγ) after stimulation with various ligands.

FIG. 16 is a graph showing that IMM70 preconditioned mice demonstratedthe highest T-cell response.

FIG. 17 is a graph showing that IMM60 caused the highest dendritic cellkilling compared to the other compounds tested.

FIG. 18 is a schematic showing the design of experiments performed toanalyze the ability of IMM47, IMM60 and IMM70 to induce T- and B-cellresponses. (Heading below: Direct T- and B-Cell Priming by IMM47, IMM60and IMM70.)

FIG. 19 is a graph analyzing tumor size in mice at various times aftertumor challenge in mice treated with indicated compounds. The data showthat IMM60 (ThrCer6) induced tumor regression.

DETAILED DESCRIPTION

Disclosed herein are compounds having a general structure of formula(I):

wherein R¹ is C₅-C₂₅ alkyl, C₅-C₂₅ alkenyl, C₅-C₂₅ alkynyl, C₅-C₂₅heteroalkyl, C₅-C₂₅ heteroalkenyl, or C₅-C₂₅ heteroalkynyl; R² and R³are each independently selected from H, OH, SH, amino or substitutedamino; R⁴ is C₅-C₂₀ alkyl, C₅-C₂₀ alkenyl, C₅-C₂₀ alkynyl, C₅-C₂₀heteroalkyl, C₅-C₂₀ heteroalkenyl, or C₅-C₂₀ heteroalkynyl; R⁶ and R⁵are each independently selected from H, alkyl, and alkenyl, or R⁶ and R⁵together form a 6-, 7-, or 8-membered cycloalkyl or cycloalkenyl ring; Xis O, S, SO₂, SO(NH), SO(N(alkyl)), NH, N(alkyl), or CH₂; Y is O, NH,N(alkyl), or S; Z is O, S, NH, N(alkyl), or CH₂; with the proviso that(a) when Y and X are each O and R⁵ and R⁶ are each H, Z is not CH₂; and(b) when Y is O, R⁶ and R⁵ together form a 6-membered cycloalkyl ring,and Z is CH₂, the cycloalkyl ring is not substituted with —CH₂OH, —OH,—CH₃, or —CH₂OCH₃, or a salt, ester, solvate, or hydrate thereof.

The compounds disclosed herein can stimulate iNKT cells. In variouscases, the compounds stimulate iNKT cells, as measured by an in vitroassay using hydridoma DN32 cells. In various cases, the compoundsstimulate iNKT cells, as measured by an in vitro assay using human iNKTcells co-cultured with C1R-hCD1d cells. In various cases, the compoundsstimulate iNKT cells in vivo.

In some embodiments, the compounds disclosed herein have a structure(IA), (IB), (IC), or (ID):

wherein n is 1, 2, or 3; m is 0, 1, or 2; p is 1 or 2; and the dashedline is an optional double bond. In some specific sets of embodiments,the dashed line is a double bond, while in other sets of embodiments,the dashed line is a single bond.

The stereochemistry of the compounds disclosed herein can be anyorientation. In some specific cases, the compounds disclosed herein havea structure of formula (IE):

Specifically excluded from the compounds of formula (I) disclosed hereinare compounds having the following structures:

Specific compounds disclosed herein include

The term “alkyl” used herein refers to a saturated or unsaturatedstraight or branched chain hydrocarbon group of one to forty carbonatoms, including, but not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like. Alkylsof one to six carbon atoms are also contemplated. The term “alkyl”includes “bridged alkyl,” i.e., a bicyclic or polycyclic hydrocarbongroup, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl,bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl. Alkylgroups optionally can be substituted, for example, with hydroxy (OH),halide, thiol (SH), aryl, heteroaryl, cycloalkyl, heterocycloalkyl, andamino. It is specifically contemplated that in the compounds describedherein the alkyl group consists of 1-40 carbon atoms, preferably 1-25carbon atoms, preferably 1-15 carbon atoms, preferably 1-12 carbonatoms, preferably 1-10 carbon atoms, preferably 1-8 carbon atoms, andpreferably 1-6 carbon atoms. A “heteroalkyl” group refers to an alkylgroup having one or more of N, S, and O.

The term “cycloalkyl” used herein refers to a hydrocarbon group arrangedin a ring. The cycloalkyl group can be substituted with one or moresubstituents, such as alkyl, halo, OH, SH, amino, substituted amino,carboxy, aryl, or heteroaryl. Examples of cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cylcononyl, and cyclodecyl. The term “heterocycloalkyl”refers to a cycloalkyl group having one or more of N, S, and O.

The term “alkenyl” used herein refers to a straight or branched chainhydrocarbon group of two to ten carbon atoms containing at least onecarbon double bond including, but not limited to, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Theterm “cycloalkenyl” refers to a cycloalkyl group having one or moredouble bonds. A “heteroalkenyl” group refers to an alkenyl group havingone or more of N, S, and O.

The term “alkynyl” used herein refers to a straight or branched chainhydrocarbon group of two to ten carbon atoms containing at least onecarbon triple bond including, but not limited to, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, and the like. A “heteroalkynyl” grouprefers to an alkynyl group having one or more of N, S, and O.

The term “alkylene” used herein refers to an alkyl group having asubstituent. For example, the term “alkylene aryl” refers to an alkylgroup substituted with an aryl group. The alkylene group is optionallysubstituted with one or more substituent previously listed as anoptional alkyl substituent. For example, an alkylene group can be—CH₂CH₂— or —CH₂—.

As used herein, the term “aryl” refers to a monocyclic or polycyclicaromatic group, preferably a monocyclic or bicyclic aromatic group,e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group canbe unsubstituted or substituted with one or more, and in particular oneto four groups independently selected from, for example, halo, alkyl,alkenyl, CF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, andheteroaryl. Exemplary aryl groups include, but are not limited to,phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl,methoxyphenyl, trifluoromethylphenyl, nitrophenyl,2,4-methoxychlorophenyl, and the like.

As used herein, the term “heteroaryl” refers to a monocyclic or bicyclicring system containing one or two aromatic rings and containing at leastone nitrogen, oxygen, or sulfur atom in an aromatic ring. Unlessotherwise indicated, a heteroaryl group can be unsubstituted orsubstituted with one or more, and in particular one to four,substituents selected from, for example, halo, alkyl, alkenyl, CF₃, NO₂,CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, and heteroaryl. In somecases, the heteroaryl group is substituted with one or more of alkyl andalkoxy groups. Examples of heteroaryl groups include, but are notlimited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl,isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl,imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, andthiadiazolyl.

The term “amino” as used herein refers to —NR₂, where R is independentlyhydrogen, optionally substituted alkyl, optionally substitutedheteroalkyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl or optionally substitutedheteroaryl. Non-limiting examples of amino groups include NH₂, NH(CH₃),and N(CH₃)₂. In some cases, R is independently hydrogen or alkyl.

The term “carboxy” or “carboxyl” used herein refers to —COOH or itsdeprotonated form —COO⁻. C₁₋₁₀carboxy refers to optionally substitutedalkyl or alkenyl groups having a carboxy moiety. Examples include, butare not limited to, —CH₂COOH, —CH₂CH(COOH)CH₃, and —CH₂CH₂CH₂COOH.

In some cases, the substituent group(s) is (are) one or more group(s)individually and independently selected from alkyl, cycloalkyl, aryl,heterocyclyl, heteroaryl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, alkoxycarbonyl, nitro,silyl, trihalomethanesulfonyl, trifluoromethyl, and amino, includingmono- and di-substituted amino groups, and the protected derivativesthereof.

Asymmetric carbon atoms can be present. All such isomers, includingdiastereomers and enantiomers, as well as the mixtures thereof, areintended to be included in the scope of the disclosure herein. Incertain cases, compounds can exist in tautomeric forms. All tautomericforms are intended to be included in the scope of the disclosure herein.Likewise, when compounds contain an alkenyl or alkenylene group, thereexists the possibility of cis- and trans-isomeric forms of thecompounds. Both cis- and trans-isomers, as well as the mixtures of cis-and trans-isomers, are contemplated.

The salts, e.g., pharmaceutically acceptable salts, of the disclosedtherapeutics may be prepared by reacting the appropriate base or acidwith a stoichiometric equivalent of the therapeutic.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, aswell as organic acids such as para-toluenesulfonic acid, salicylic acid,tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylicacid, fumaric acid, gluconic acid, glucuronic acid, formic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonicacid, carbonic acid, succinic acid, citric acid, benzoic acid and aceticacid, as well as related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, O-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and othersalts. In one embodiment, pharmaceutically acceptable acid additionsalts include those formed with mineral acids such as hydrochloric acidand hydrobromic acid, and especially those formed with organic acidssuch as maleic acid.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.Examples of metals used as cations are sodium, potassium, magnesium,ammonium, calcium, or ferric, and the like. Examples of suitable aminesinclude isopropylamine, trimethylamine, histidine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

Similarly, pharmaceutically acceptable derivatives (e.g., esters),metabolites, hydrates, solvates and prodrugs of the therapeutic may beprepared by methods generally known to those skilled in the art. Thus,another embodiment provides compounds that are prodrugs of an activecompound. In general, a prodrug is a compound which is metabolized invivo (e.g., by a metabolic transformation such as deamination,dealkylation, de-esterification, and the like) to provide an activecompound. A “pharmaceutically acceptable prodrug” means a compound whichis, within the scope of sound medical judgment, suitable forpharmaceutical use in a patient without undue toxicity, irritation,allergic response, and the like, and effective for the intended use,including a pharmaceutically acceptable ester as well as a zwitterionicform, where possible, of the therapeutic. As used herein, the term“pharmaceutically acceptable ester” refers to esters that hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Suitable ester groupsinclude, for example, those derived from pharmaceutically acceptablealiphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Representativeexamples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.Examples of pharmaceutically-acceptable prodrug types are described inHiguchi and Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of theA.C.S. Symposium Series, and in Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference.

The compounds and compositions described herein may also includemetabolites. As used herein, the term “metabolite” means a product ofmetabolism of a compound of the embodiments or a pharmaceuticallyacceptable salt, analog, or derivative thereof, that exhibits a similaractivity in vitro or in vivo to a disclosed therapeutic. The compoundsand compositions described herein may also include hydrates andsolvates. As used herein, the term “solvate” refers to a complex formedby a solute (herein, the therapeutic) and a solvent. Such solvents forthe purpose of the embodiments preferably should not negativelyinterfere with the biological activity of the solute. Solvents may be,by way of example, water, ethanol, or acetic acid.

Synthesis of Compounds

The compounds described herein can be synthesized using any means knownto the synthetic organic chemist. Described below are synthetic schemesfor synthesizing several of the compounds as disclosed herein, and canbe used as guidance on synthesis of the compounds disclosed herein.

The completely selective formation of α-galactosides has traditionallybeen difficult; however in recent years some excellent solutions to thisproblem have been developed. The Nishida and Kobayashi's dehydrativeglycosylation methodology was employed; thus reaction of2,3,4,6-tetra-O-benzyl-galactose 14 with CBr₄/PPh₃ afforded thecorresponding galactosyl bromide, which was reacted in situ withacceptor 15, in the presence of tetramethylurea (TMU) and Bu₄NBr, toprovide a good yield of the desired galactoside 16 as a single α-anomer(Scheme 1). Staudinger reduction of the azide in 16 with PMe₃ in wet THFafforded amine 17, which reacted with hexacosanoyl chloride to provideamide 18 in an unoptimized 40% yield from azide 16. Formation of thecorresponding urea 19 from amine 17 required the synthesis of anappropriate isocyanate, formed by a Curtius rearrangement on thecorresponding acid azide. Use of hexacosanoic acid as the starting pointwould lead to a urea product containing 27 atoms (26 carbons and onenitrogen) in the acyl chain. The hydrophobic A′ binding pocket in CD1doptimally accommodates an acyl chain length containing 26 carbon atoms.So, tetracosanoic acid was used as this would be processed through to aurea product containing 25 atoms in the acyl chain (24 carbons and onenitrogen). Since the α-GalCer analogue containing a C₂₄ acyl chaindisplays similar biological activity to α-GalCer containing a C₂₆ chain,differences in biological activity between a ureido analogue containing25 atoms in the acyl chain (i.e. 7), and α-GalCer would be aattributable to an amide-urea switch and not a result of the slightlytruncated alkyl chain length. Tricosanyl isocyanate was duly preparedfrom tetracosanoic acid in three steps. Treatment of tetracosanoylchloride with NaN₃ afforded the corresponding acid azide, whichunderwent Curtius rearrangement on heating in toluene at reflux toprovide tricosanyl isocyanate. Without purification, the isocyanate wasreacted with amine 17 to provide urea 19 in 68% yield. Hydrogenolysis ofthe benzyl groups in amide 18 and urea 19 effected global deprotectionand afforded urea 7, alongside α-GalCer 1, which would serve as thecontrol in the biological studies (Scheme 1).

Synthesis of α-GalCer 1 and urea 7. (a) 14, PPh₃, CBr₄, CH₂Cl₂, r.t., 3h; then Me₂C(O)NMe₂, Bu₄NBr, CH₂Cl₂; then 15, CH₂Cl₂, 3 Å MS, r.t., 3 d,62%. (b) PMe₃, THF, r.t., 4 h, then H₂O, 1 h, 72%. (c) CH₃(CH₂)₂₄C(O)Cl,Et₃N, CH₂Cl₂, 0° C. to r.t., 8 h, 18 (54%). (d) CH₃(CH₂)₂₂NCO, toluene,reflux, 8 h, 19 (68%). (e) Pd(OH)₂/C, H₂, THF, r.t., 22 h: 1 (68% from17); 7 (73% from 17).

While benzyl ethers are commonly employed protecting groups,particularly in carbohydrate chemistry, conformational effects can meanthat some groups are particularly stubborn to remove. Indeed, thisproved to be the case with one of the benzyl ethers in thephytosphingosine unit of amide 18 and urea 19; debenzylation was oftenslow, and frequently required filtration of the reaction mixture andaddition of fresh catalyst to effect complete deprotection. To addressthis troublesome issue, a modified phytosphingosine acceptor wasinvestigated in which the internal 1,2-diol was protected as anisopropylidene acetal. The use of an acetal to protect the internal1,2-diol in phytosphingosine would likely necessitate a two-stepdeprotection post glycosylation; however a late-stage acetal hydrolysiswas not problematic, and more importantly, the debenzylation step wouldbe significantly easier. Moreover the additional deprotection step wouldbe mitigated by its straightforward two-step synthesis fromphytosphingosine, compared with the three-step synthesis required toaccess dibenzyl ether 15.

Although the choice of donor/acceptor pairs can impact on thestereoselectivity of glycosylation reactions, galactoside 20 could beaccessed under standard conditions in similarly good yield and onceagain with complete α-stereoselectivity (Scheme 2). SubsequentStaudinger reduction provided amine 21, which was acylated as before toprovide amide 22. Alternatively, reaction with a mixed carbonate,prepared from 1-tetracosanol and N,N′-disuccinimidyl carbonate providedcarbamate 23. A two-step acetal hydrolysis/debenzylation sequence on 22and 23 proceeded uneventfully in both cases, to provide α-GalCer andcarbamate derivative 8, respectively. Finally the thioamide 6 wasprepared from α-GalCer in a three-step sequence, involvingperacetylation to provide 24, chemoselective thionation of the amidewith Lawesson's reagent to afford thioamide 25, followed bydeacetylation under Zémplen conditions (Scheme 2).

Improved synthesis of α-GalCer 1, and synthesis of carbamate 8 andthioamide 6. (a) PMe₃, THF, 3 h, r.t., then H2O, 1 h, 93%. (b)CH₃(CH₂)₂₄C(O)Cl, Et₃N, CH₂Cl₂, 0° C. to r.t., 12 h, 22 (85%). (c)N-succinimidyl-tetracosanyl carbonate, Et₃N, CH₂Cl₂, r.t., 4 h, 23(82%). (d) from 22: (i) TFA, CH₂Cl₂—H₂O, 10:1, 2 h, r.t.; (ii)Pd(OH)₂/C, H₂, THF, 6 h, 1 (75%). (e) from 23: (i) TFA, CH₂Cl₂—MeOH,2:1, 2 h, r.t.; (ii) Pd(OH)₂/C, H₂, THF, 6 h, 8 (75%). (f) Ac₂O,pyridine, r.t., 10 h, 94%. (g) Lawesson's reagent, toluene, 80° C., 4 h,85%. (h) NaOMe, MeOH, r.t., 2 h, 90%.

Synthesis of ThrCer 2 and its three analogues 9, 10 and 11 are describednext. Ready access to an advanced intermediate, namely amine 26, using aslight modification of the previously established methodology alongsidethat developed for generating the three α-GalCer analogues, providedstraightforward access to the corresponding ThrCer analogues assummarized in Scheme 3. ThrCer 2 was synthesized from amine 26 in athree-step sequence involving acylation, followed by silyl etherdeprotection and acetal hydrolysis. Thionation of the acylation product27 provided thioamide 28, which underwent the same two deprotectionsteps to afford the first ThrCer target, namely thioamide analogue 9.Alternatively, treatment of amine 26 with the mixed carbonate derivedfrom the reaction of 1-tetracosanol with N,N′-disuccinimidyl carbonate,provided carbamate 29, and with tricosanyl isocyanate, furnished urea30, and thence the final two targets, carbamate 11 and urea 10, aftersilyl deprotection and acetal hydrolysis (Scheme 3).

Synthesis of ThrCer 2 and thioamide, urea and carbamate analogues. (a)CH₃(CH₂)₂₄C(O)Cl, Et₃N, CH₂Cl₂, 0° C. to r.t., 12 h, 85%. (b) Lawesson'sreagent, toluene, 80° C., 5 h, 88%. (c) Bu₄NF, THF, r.t., 4 h. (d) TFA,CH₂Cl₂ ^(—)MeOH (10:1), r.t.; 2 (74% from 27); 9 (73% from 28); 10 (72%from 30); 11 (70% from 29). (e) N-succinimidyl-tetracosanyl carbonate,Et₃N, CH₂Cl₂, r.t., 5 h, 29 (86%). (f) CH₃(CH₂)₂₄NCO, toluene, reflux, 8h, 30 (80%).

Scheme 4 shows a general synthesis for preparing sulfamidate compoundsas disclosed herein.

Scheme 5 shows a general synthesis for preparing seven-membered ringcompounds as disclosed herein.

Scheme 6 shows a general synthesis for preparing six-membered ringcompounds as disclosed herein.

Scheme 7 shows a general synthesis for preparing eight-membered ringcompounds as disclosed herein.

Pharmaceutical Formulations and Routes of Administration

As herein, the compounds described herein may be formulated inpharmaceutical compositions with a pharmaceutically acceptableexcipient. The compound or composition comprising the compound isadministered by any route that permits treatment of the disease orcondition.

One route of administration is oral administration. Additionally, thecompound or composition comprising the compound may be delivered to apatient using any standard route of administration, includingparenterally, such as intravenously, intraperitoneally, intrapulmonary,subcutaneously or intramuscularly, intrathecally, topically,transdermally, rectally, orally, nasally or by inhalation. Slow releaseformulations may also be prepared from the agents described herein inorder to achieve a controlled release of the active agent in contactwith the body fluids in the gastro intestinal tract, and to provide asubstantial constant and effective level of the active agent in theblood plasma. The crystal form may be embedded for this purpose in apolymer matrix of a biological degradable polymer, a water-solublepolymer or a mixture of both, and optionally suitable surfactants.Embedding can mean in this context the incorporation of micro-particlesin a matrix of polymers. Controlled release formulations are alsoobtained through encapsulation of dispersed micro-particles oremulsified micro-droplets via known dispersion or emulsion coatingtechnologies.

Administration may take the form of single dose administration, or acompound as disclosed herein can be administered over a period of time,either in divided doses or in a continuous-release formulation oradministration method (e.g., a pump). However the compounds of theembodiments are administered to the subject, the amounts of compoundadministered and the route of administration chosen should be selectedto permit efficacious treatment of the disease condition.

In an embodiment, the pharmaceutical compositions are formulated withone or more pharmaceutically acceptable excipient, such as carriers,solvents, stabilizers, adjuvants, diluents, etc., depending upon theparticular mode of administration and dosage form. The pharmaceuticalcompositions should generally be formulated to achieve a physiologicallycompatible pH, and may range from a pH of about 3 to a pH of about 11,preferably about pH 3 to about pH 7, depending on the formulation androute of administration. In alternative embodiments, the pH is adjustedto a range from about pH 5.0 to about pH 8. More particularly, thepharmaceutical compositions may comprise a therapeutically orprophylactically effective amount of at least one compound as describedherein, together with one or more pharmaceutically acceptableexcipients. Optionally, the pharmaceutical compositions may comprise acombination of the compounds described herein, or may include a secondactive ingredient useful in the treatment or prevention of bacterialinfection (e.g., anti-bacterial or anti-microbial agents).

Formulations, e.g., for parenteral or oral administration, are mosttypically solids, liquid solutions, emulsions or suspensions, whileinhalable formulations for pulmonary administration are generallyliquids or powders. A pharmaceutical composition can also be formulatedas a lyophilized solid that is reconstituted with a physiologicallycompatible solvent prior to administration. Alternative pharmaceuticalcompositions may be formulated as syrups, creams, ointments, tablets,and the like.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compoundsdescribed herein. The term refers to any pharmaceutical excipient thatmay be administered without undue toxicity.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there exists awide variety of suitable formulations of pharmaceutical compositions(see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA),carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and/orhydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oils, water,saline, glycerol and/or ethanol) wetting or emulsifying agents, pHbuffering substances, and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions described herein are formulated in anyform suitable for an intended method of administration. When intendedfor oral use for example, tablets, troches, lozenges, aqueous or oilsuspensions, non-aqueous solutions, dispersible powders or granules(including micronized particles or nanoparticles), emulsions, hard orsoft capsules, syrups or elixirs may be prepared. Compositions intendedfor oral use may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as cross-linked povidone, maizestarch, or alginic acid; binding agents, such as povidone, starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc.

Tablets may be uncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions may be formulated assuspensions comprising a compound of the embodiments in admixture withat least one pharmaceutically acceptable excipient suitable for themanufacture of a suspension.

In yet another embodiment, pharmaceutical compositions may be formulatedas dispersible powders and granules suitable for preparation of asuspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents (e.g., sodium carboxymethylcellulose, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth, gum acacia); dispersing or wetting agents (e.g., anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycethanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate)); andthickening agents (e.g., carbomer, beeswax, hard paraffin or cetylalcohol). The suspensions may also contain one or more preservatives(e.g., acetic acid, methyl or n-propyl p-hydroxy-benzoate); one or morecoloring agents; one or more flavoring agents; and one or moresweetening agents such as sucrose or saccharin.

The pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth; naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids; hexitol anhydrides, such as sorbitan monooleate; and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan monooleate. The emulsion may also containsweetening and flavoring agents. Syrups and elixirs may be formulatedwith sweetening agents, such as glycerol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, a flavoringor a coloring agent.

Additionally, the pharmaceutical compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated by a person of ordinary skill in the art using those suitabledispersing or wetting agents and suspending agents, including thosementioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids (e.g., oleicacid) may likewise be used in the preparation of injectables.

To obtain a stable water-soluble dose form of a pharmaceuticalcomposition, a pharmaceutically acceptable salt of a compound describedherein may be dissolved in an aqueous solution of an organic orinorganic acid, such as 0.3 M solution of succinic acid, or morepreferably, citric acid. If a soluble salt form is not available, thecompound may be dissolved in a suitable co-solvent or combination ofco-solvents. Examples of suitable co-solvents include alcohol, propyleneglycol, polyethylene glycol 300, polysorbate 80, glycerin and the likein concentrations ranging from about 0 to about 60% of the total volume.In one embodiment, the active compound is dissolved in DMSO and dilutedwith water.

The pharmaceutical composition may also be in the form of a solution ofa salt form of the active ingredient in an appropriate aqueous vehicle,such as water or isotonic saline or dextrose solution. Also contemplatedare compounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In some embodiments, the compounds described herein may be formulatedfor oral administration in a lipid-based formulation suitable for lowsolubility compounds. Lipid-based formulations can generally enhance theoral bioavailability of such compounds.

As such, pharmaceutical compositions comprise a therapeutically orprophylactically effective amount of a compound described herein,together with at least one pharmaceutically acceptable excipientselected from the group consisting of medium chain fatty acids andpropylene glycol esters thereof (e.g., propylene glycol esters of ediblefatty acids, such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants, such as polyoxyl 40hydrogenated castor oil.

In some embodiments, cyclodextrins may be added as aqueous solubilityenhancers. Exemplary cyclodextrins include hydroxypropyl, hydroxyethyl,glucosyl, maltosyl and maltotriosyl derivatives of α-, β-, andγ-cyclodextrin. A specific cyclodextrin solubility enhancer ishydroxypropyl-o-cyclodextrin (BPBC), which may be added to any of theabove-described compositions to further improve the aqueous solubilitycharacteristics of the compounds of the embodiments. In one embodiment,the composition comprises about 0.1% to about 20%hydroxypropyl-o-cyclodextrin, more preferably about 1% to about 15%hydroxypropyl-o-cyclodextrin, and even more preferably from about 2.5%to about 10% hydroxypropyl-o-cyclodextrin. The amount of solubilityenhancer employed will depend on the amount of the compound of theinvention in the composition.

Methods of Treatment

Provided herein are methods of different types of cancer in a subject(e.g., a mammal) in need thereof comprising administering to the subjectthe compound or composition as described herein in an amount effectiveto treat said cancer. In some cases, the mammalian subject is a humansubject. Practice of methods described herein in other mammaliansubjects, especially mammals that are conventionally used as models fordemonstrating therapeutic efficacy in humans (e.g., primate, porcine,canine, or rabbit animals), is also contemplated. Standard dose-responsestudies are used to optimize dose and dosing schedule.

The disclosed methods are useful for treating cancer, for example,inhibiting cancer growth, including complete cancer remission, forinhibiting cancer metastasis, and for promoting cancer resistance. Theterm “cancer growth” generally refers to any one of a number of indicesthat suggest change within the cancer to a more developed form. Thus,indices for measuring an inhibition of cancer growth include but are notlimited to a decrease in cancer cell survival, a decrease in tumorvolume or morphology (for example, as determined using computedtomographic (CT), sonography, or other imaging method), a delayed tumorgrowth, a destruction of tumor vasculature, improved performance indelayed hypersensitivity skin test, an increase in the activity ofcytolytic T-lymphocytes, and a decrease in levels of tumor-specificantigens.

The term “cancer resistance” refers to an improved capacity of a subjectto resist cancer growth, in particular growth of a cancer already had.In other words, the term “cancer resistance” refers to a decreasedpropensity for cancer growth in a subject.

In one aspect, the cancer comprises a solid tumor, for example, acarcinoma and a sarcoma. Carcinomas include malignant neoplasms derivedfrom epithelial cells which infiltrate, for example, invade, surroundingtissues and give rise to metastases. Adenocarcinomas are carcinomasderived from glandular tissue, or from tissues that form recognizableglandular structures. Another broad category of cancers includessarcomas and fibrosarcomas, which are tumors whose cells are embedded ina fibrillar or homogeneous substance, such as embryonic connectivetissue. The invention also provides methods of treatment of cancers ofmyeloid or lymphoid systems, including leukemias, lymphomas, and othercancers that typically are not present as a tumor mass, but aredistributed in the vascular or lymphoreticular systems. Furthercontemplated are methods for treatment of adult and pediatric oncology,growth of solid tumors/malignancies, myxoid and round cell carcinoma,locally advanced tumors, cancer metastases, including lymphaticmetastases. The cancers listed herein are not intended to be limiting.Age (child and adult), sex (male and female), primary and secondary,pre- and post-metastatic, acute and chronic, benign and malignant,anatomical location cancer embodiments and variations are contemplatedtargets. Cancers are grouped by embryonic origin (e.g., carcinoma,lymphomas, and sarcomas), by organ or physiological system, and bymiscellaneous grouping. Particular cancers may overlap in theirclassification, and their listing in one group does not exclude themfrom another.

Carcinomas that may targeted include adrenocortical, acinar, aciniccell, acinous, adenocystic, adenoid cystic, adenoid squamous cell,cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex,adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar,alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroidgland, apocrine, basal cell, basal cell, alveolar, comedo basal cell,cystic basal cell, morphea-like basal cell, multicentric basal cell,nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basalcell, superficial basal cell, basaloid, basosquamous cell, bile duct,extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar,bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar cell,bronchogenic, cerebriform, cholangiocelluarl, chorionic, choroidsplexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer ofcorpus uteri, cortisol-producing, cribriform, cylindrical, cylindricalcell, duct, ductal, ductal cancer of the prostate, ductal cancer in situ(DCIS), eccrine, embryonal, cancer en cuirasse, endometrial, cancer ofendometrium, endometroid, epidermoid, cancer ex mixed tumor, cancer expleomorphic adenoma, exophytic, fibrolamellar, cancer fibro'sum,follicular cancer of thyroid gland, gastric, gelatinform, gelatinous,giant cell, giant cell cancer of thyroid gland, cancer gigantocellulare,glandular, granulose cell, hepatocellular, Wirthle cell, hypernephroid,infantile embryonal, islet cell carcinoma, inflammatory cancer of thebreast, cancer in situ, intraductal, intraepidermal, intraepithelial,juvenile embryonal, Kulchitsky-cell, large cell, leptomeningeal,lobular, infiltrating lobular, invasive lobular, lobular cancer in situ(LCIS), lymphoepithelial, cancer medullare, medullary, medullary cancerof thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell,metatypical cell, micropapillary, mucinous, cancer muciparum, cancermucocellulare, mucoepidermoid, cancer mucosum, mucous, nasopharyngeal,neuroendocrine cancer of the skin, noninfiltrating, non-small cell,non-small cell lung cancer (NSCLC), oat cell, cancer ossificans,osteoid, Paget's, papillary, papillary cancer of thyroid gland,periampullary, preinvasive, prickle cell, primary intrasseous, renalcell, scar, schistosomal bladder, Schneiderian, scirrhous, sebaceous,signet-ring cell, cancer simplex, small cell, small cell lung cancer(SCLC), spindle cell, cancer spongiosum, squamous, squamous cell,terminal duct, anaplastic thyroid, follicular thyroid, medullarythyroid, papillary thyroid, trabecular cancer of the skin, transitionalcell, tubular, undifferentiated cancer of thyroid gland, uterine corpus,verrucous, villous, cancer villosum, yolk sac, squamous cellparticularly of the head and neck, esophageal squamous cell, and oralcancers and carcinomas.

Sarcomas that may be targeted include adipose, alveolar soft part,ameloblastic, avian, botryoid, sarcoma botryoides, chicken,chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal,endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl,giant cell, granulocytic, hemangioendothelial, Hodgkin's, idiopathicmultiple pigmented hemorrhagic, immunoblastic sarcoma of B cells,immunoblastic sarcoma of T cells, Jensen's, Kaposi's, kupffer cell,leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio,idiopathic hemorrhagic, multipotential primary sarcoma of bone,osteoblastic, osteogenic, parosteal, polymorphous, pseudo-kaposi,reticulum cell, reticulum cell sarcoma of the brain, rhabdomyosarcoma,rous, soft tissue, spindle cell, synovial, telangiectatic, sarcoma(osteosarcoma)/malignant fibrous histiocytoma of bone, and soft tissuesarcomas.

Lymphomas that may be targeted include AIDS-related, non-Hodgkin's,Hodgkin's, T-cell, T-cell leukemia/lymphoma, African, B-cell, B-cellmonocytoid, bovine malignant, Burkitt's, centrocytic, lymphoma cutis,diffuse, diffuse, large cell, diffuse, mixed small and large cell,diffuse, small cleaved cell, follicular, follicular center cell,follicular, mixed small cleaved and large cell, follicular,predominantly large cell, follicular, predominantly small cleaved cell,giant follicle, giant follicular, granulomatous, histiocytic, largecell, immunoblastic, large cleaved cell, large nocleaved cell,Lennert's, lymphoblastic, lymphocytic, intermediate; lymphocytic,intermediately differentiated, plasmacytoid; poorly differentiatedlymphocytic, small lymphocytic, well differentiated lymphocytic,lymphoma of cattle; MALT, mantle cell, mantle zone, marginal zone,Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular,plasmacytoid, pleomorphic, primary central nervous system, primaryeffusion, small b-cell, small cleaved cell, small concleaved cell,T-cell lymphomas; convoluted T-cell, cutaneous t-cell, small lymphocyticT-cell, undefined lymphoma, u-cell, undifferentiated, aids-related,central nervous system, cutaneous T-cell, effusion (body cavity based),thymic lymphoma, and cutaneous T cell lymphomas.

Leukemias and other blood cell malignancies that may be targeted includeacute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenousleukemia, chronic myelogenous, hairy cell, erythroleukemia,lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia, hairy cell,T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell,basophilic, hemoblastic, histiocytic, leukopenic, lymphatic,Schilling's, stem cell, myelomonocytic, monocytic, prolymphocytic,promyelocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic,rieder cell, bovine, aleukemic, mast cell, myelocytic, plamsa cell,subleukemic, multiple myeloma, nonlymphocytic, chronic myelogenousleukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma,Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high gradeforms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease, myelodysplastic syndrome, and myelodysplasia and chronicmyelocytic leukemias.

Brain and central nervous system (CNS) cancers and tumors that may betargeted include astrocytomas (including cerebellar and cerebral), brainstem glioma, brain tumors, malignant gliomas, ependymoma, glioblastoma,medulloblastoma, supratentorial primitive neuroectodermal tumors, visualpathway and hypothalamic gliomas, primary central nervous systemlymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamicglioma, extracranial germ cell tumor, medulloblastoma, myelodysplasticsyndromes, oligodendroglioma, myelodysplastic/myeloproliferativediseases, myelogenous leukemia, myeloid leukemia, multiple myeloma,myeloproliferative disorders, neuroblastoma, plasma cellneoplasm/multiple myeloma, central nervous system lymphoma, intrinsicbrain tumors, astrocytic brain tumors, gliomas, and metastatic tumorcell invasion in the central nervous system.

Gastrointestimal cancers that may be targeted include extrahepatic bileduct cancer, colon cancer, colon and rectum cancer, colorectal cancer,gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoidtumor, gastronintestinal carcinoid tumors, gastrointestinal stromaltumors, bladder cancers, islet cell carcinoma (endocrine pancreas),pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectalcancer, salivary gland cancer, small intestine cancer, colon cancer, andpolyps associated with colorectal neoplasia.

Lung and respiratory cancers that may be targeted include bronchialadenomas/carcinoids, esophagus cancer esophageal cancer, esophagealcancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer,lung carcinoid tumor, non-small cell lung cancer, small cell lungcancer, small cell carcinoma of the lungs, mesothelioma, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, nasopharyngealcancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer;paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.

Urinary tract and reproductive cancers that may be targeted includecervical cancer, endometrial cancer, ovarian epithelial cancer,extragonadal germ cell tumor, extracranial germ cell tumor, extragonadalgerm cell tumor, ovarian germ cell tumor, gestational trophoblastictumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer,ovarian germ cell tumor, ovarian low malignant potential tumor, penilecancer, renal cell cancer (including carcinomas), renal cell cancer,renal pelvis and ureter (transitional cell cancer), transitional cellcancer of the renal pelvis and ureter, gestational trophoblastic tumor,testicular cancer, ureter and renal pelvis, transitional cell cancer,urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginalcancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelialneoplasms, cervical carcinoma, uterine cancer and solid tumors in theovarian follicle), superficial bladder tumors, invasive transitionalcell carcinoma of the bladder, and muscle-invasive bladder cancer.

Skin cancers and melanomas (as well as non-melanomas) that may betargeted include cutaneous t-cell lymphoma, intraocular melanoma, tumorprogression of human skin keratinocytes, basal cell carcinoma, andsquamous cell cancer. Liver cancers that may be targeted includeextrahepatic bile duct cancer, and hepatocellular cancers. Eye cancersthat may be targeted include intraocular melanoma, retinoblastoma, andintraocular melanoma Hormonal cancers that may be targeted include:parathyroid cancer, pineal and supratentorial primitive neuroectodermaltumors, pituitary tumor, thymoma and thymic carcinoma, thymoma, thymuscancer, thyroid cancer, cancer of the adrenal cortex, and ACTH-producingtumors.

Miscellaneous other cancers that may be targeted include advancedcancers, AIDS-related, anal cancer adrenal cortical, aplastic anemia,aniline, betel, buyo cheek, cerebriform, chimney-sweeps, clay pipe,colloid, contact, cystic, dendritic, cancer à deux, duct, dye workers,encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial,glandular, cancer in situ, kang, kangri, latent, medullary, melanotic,mule-spinners', non-small cell lung, occult cancer, paraffin, pitchworkers', scar, schistosomal bladder, scirrhous, lymph node, small celllung, soft, soot, spindle cell, swamp, tar, and tubular cancers.

Miscellaneous other cancers that may be targeted also include carcinoid(gastrointestinal and bronchal) Castleman's disease chronicmyeloproliferative disorders, clear cell sarcoma of tendon sheaths,Ewing's family of tumors, head and neck cancer, lip and oral cavitycancer, Waldenstrom's macroglobulinemia, metastatic squamous neck cancerwith occult primary, multiple endocrine neoplasia syndrome, multiplemyeloma/plasma cell neoplasm, Wilms' tumor, mycosis fungoides,pheochromocytoma, sezary syndrome, supratentorial primitiveneuroectodermal tumors, unknown primary site, peritoneal effusion,malignant pleural effusion, trophoblastic neo-plasms, andhemangiopericytoma.

Further described herein are methods of stimulating an immune responsein a mammalian subject comprising administering to the subject acompound or composition described herein. In some embodiments, thecompound or composition is administered directly to the subject in thesame manner as a vaccine. In some embodiments, the compounds describedherein are useful for the induction of an immune response to a tumorantigen, one or more pathogenic organisms, or other antigen as describedherein.

Dosing

The terms “therapeutically effective amount” and “prophylacticallyeffective amount,” as used herein, refer to an amount of a compoundsufficient to treat, ameliorate, or prevent the identified disease orcondition, or to exhibit a detectable therapeutic, prophylactic, orinhibitory effect. The effect can be detected by, for example, animprovement in clinical condition, reduction in symptoms, or by any ofthe assays or clinical diagnostic tests described herein. The preciseeffective amount for a subject will depend upon the subject's bodyweight, size, and health; the nature and extent of the condition; andthe therapeutic or combination of therapeutics selected foradministration. Therapeutically and prophylactically effective amountsfor a given situation can be determined by routine experimentation thatis within the skill and judgment of the clinician.

Dosages of the therapeutic can be administered as a dose measured in mg.Contemplated dosages of the disclosed therapeutics include about 0.1 mgto 5000 mg (5 g). Specific ranges of doses in mg include about 1 mg toabout 4000 mg, about 2 mg to about 3000 mg, about 5 mg to about 2000 mg,about 5 mg to about 1000 mg, about 10 mg to about 1000 mg, about 20 mgto about 500 mg, about 30 mg to about 200 mg, and about 50 mg to about100 mg. The doses can be total daily amounts given to a subject or thedose given at any single time. Thus, the dose can be administered as asingle dose or in divided doses throughout the day (e.g., in two, three,four, or five doses over the course of a day).

Dosages of the therapeutic can alternately be administered as a dosemeasured in mg/kg (mg compound per kilogram of body weight for thetreated subject). Contemplated mg/kg doses of the disclosed therapeuticsinclude about 0.001 mg/kg to about 1000 mg/kg. Specific ranges of dosesin mg/kg include about 0.1 mg/kg to about 500 mg/kg, about 0.5 mg/kg toabout 200 mg/kg, about 1 mg/kg to about 100 mg/kg, about 2 mg/kg toabout 50 mg/kg, and about 5 mg/kg to about 30 mg/kg.

Combination Therapy

The methods disclosed herein can also include the use of a compound orcompounds as described herein together with one or more additionaltherapeutic agents for the treatment of disease conditions. Thus, forexample, the combination of active ingredients may be: (1) co-formulatedand administered or delivered simultaneously in a combined formulation;(2) delivered by alternation or in parallel as separate formulations; or(3) by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods described herein maycomprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used. In some cases, a compounddisclosed herein is administered and/or formulated with a secondtherapeutic.

The second therapeutic can be one or more of a chemotherapeutic or animmunotherapeutic agent. In some specific cases, the second therapeuticis a cytokine, an anti-inflammatory agent, a cancer vaccine, a cancerantigen, or a polynuecleotide encoding a cancer antigen. In some cases,the second therapeutic is radiation.

Contemplated chemotherapeutics for use in combination therapies asdisclosed herein include aspirin, sulindac, curcumin, alkylating agentsincluding: nitrogen mustards, such as mechlor-ethamine,cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas,such as carmustine (BCNU), lomustine (CCNU), and semustine(methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine(TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine(HMM, altretamine); alkyl sulfonates such as busulfan; triazines such asdacarbazine (DTIC); antimetabolites including folic acid analogs such asmethotrexate and trimetrexate, pyrimidine analogs such as5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside(AraC, cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purineanalogs such as 6-mercaptopurine, 6-thioguanine, azathioprine,2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA),fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA);natural products including antimitotic drugs such as paclitaxel, vincaalkaloids including vinblastine (VLB), vincristine, and vinorelbine,taxotere, estramustine, and estramustine phosphate; epipodophylotoxinssuch as etoposide and teniposide; antibiotics such as actimomycin D,daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin,bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; andenzymes such as L-asparaginase.

Contemplated biological response modifying agents for use in combinationtherapies as disclosed herein include, but are not limited to,interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents includingplatinum coordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; non-steroidal antiandrogens such as flutamide; kinaseinhibitors, histone deacetylase inhibitors, methylation inhibitors,proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants,telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, statinhibitors and receptor tyrosin kinase inhibitors such as imatinibmesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptorinhibitor) now marketed as Tarveca; and anti-virals such as oseltamivirphosphate, Amphotericin B, and palivizumab.

Contemplated immunotherapeutic agents for use in the combinationtherapies disclosed herein include, but are not limited to a Her2/neureceptor antibody such as trastuzumab (marketed as Herceptin®), ananti-CD52 antibody such as alemtuzumab (marketed as Campath®.MabCampath® or Campath-1H), an anti-CD33 antibody such as gemtuzumablinked to calicheamicin (marketed as Mylotarg®), an anti-CD20 antibodysuch as rituximab (marketed as Rituxan® and MabThera®), Ibritumomabtiuxetan (marketed as Zevalin®), anti-TNFα antibodies such as infliximab(marketed as Remicade®) or adalimmumab (marketed as Humira®), a solubleTNFR2 molscule such as etanercept (marketed as Enbrel®), an antibody tothe CD25 chain of the IL-2 receptor such as basiliximab (marketed asSimulect®), an anti CD40/CD40L antibody such as humanized IgG1anti-human CD40 antibody (SGN-40), an anti-CTLA-4 blocking antibody suchas iplimumab (marketed as MDX-101 or MDX-010) or tremelimumab, ananti-programmed death protein 1 (PD-1) antibody (i.e., an anti-CD279antibody), an anti-programmed cell death ligand (PDL-1) antibody, ananti-glucocorticoid-induced TNFR-related gene (GITR) antibody, ananti-OX-40 (CD134) antibody, soluble lymphocyte-activation gene 3 (alsoknown asLAG3 or CD223)-based immune modulator such as LAG3-Ig (IMP321),Toll-like receptor agonists such as monophosphoril lipid A (MPL®), CpG,single-stranded RNA, nucleotides, nucleotide analogue, CL087 (aTLR7-specific ligand), loxoribine, polyinosine-polycytidylic acid,flagellin, resiquimod, immiquimod, gardiquimod, NOD ligands such asmuramyl dipeptide, murabutide, peptidoglycan and muramyldipeptide,

In some embodiments, a combination therapy as disclosed herein comprisesadministration of a compound disclosed herein, such as a compound havinga structure:

wherein n is 1, 2, or 3, or a salt, ester, solvate, or hydrate thereofand one or more antibodies selected from the group consisting of ananti-PD1 antibody, an anti-PDL-1 antibody, an anti-CTLA-4 antibody ananti-GITR antibody and an anti-OX40 antibody.

In some embodiments, the anti-PD1 antibody is a monoclonal antibodydirected against the negative immynoregulatory human cell surfacereceptor PD-1 with immunopotentiation activity. An exemplary anti-PD1antibody is human monoclonal antibody MDX-1106 which binds and blocksthe activation of PD-1 by its ligands PD-L1 and PD-L2, resulting in theactivation of T-cells and cell-mediated immune responses against tumorcells. In some embodiments, the anti-PD-L1 antibody is a monoclonalantibody directed against the protein ligand PD-L1 with immunomodulatingand antineoplastic activities. An exemplary anti-PD-L1 antibody is humanmonoclonal antibody MDX-1105 which binds PD-L1 and blocks its binding toand activation of its receptor PD-1, which may enhance theT-cell-mediated immune response to neoplasms and reverse T-cellinactivation in chronic infections disease. PD-L1 is expressed broadlyon hematopoietic and parenchymal tissues.

In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibodydirected against the T-cell receptor protein cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). An exemplary anti-CTLA-4antibody is human IgG2 monoclonal antibody tremelimumab which binds toCTLA4 and blocks binding of the antigen presenting cell ligands B7-1 andB7-2 to CTLA-4, resulting in inhibition of B7-CTLA4-mediateddownregulation of T-cell activation. Another exemplary anti-CTLA-4antibody is human IgG1 monoclonal antibody ipilimumab which binds toCTLA4 and blocks binding of the antigen presenting cell ligands B7-1 andB7-2 to CTLA-4, resulting in inhibition of B7-CTLA4-mediateddownregulation of T-cell activation. Ipilimumab is undergoing clinicaltrials for the treatment of non-small cell lung carcinoma, small celllung cancer and metastatic hormone-refractory prostate cancer.

In some embodiments, the anti-GITR antibody is a monoclonal antibodydirected against glucocorticoid-induced tumor necrosis factor receptor(GITR) which blocks the interaction of GITR with its ligand, enhancescytotoxicity of natural human killer cells and/or downmodulates GITRexpression on peripheral blood lymphocytes.

In some embodiments, the an anti-OX40 antibody is an agonisticmonoclonal antibody that mimicks the natural OX40 ligand and selectivelybinds to and activates the OX40 receptor. Receptor activation inducesproliferation of memory and effector T cells

Cytokines that are effective in inhibiting tumor growth/metastasis arecontemplated for use in the combination therapy. Such cytokines,lymphokines, or other hematopoietic factors include, but are not limitedto, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cellfactor, and erythropoietin.

An immunotherapeutic agent can be a cancer vaccine. A cancer vaccine isan agent, molecule, or immunogen which stimulates or elicits anendogenous immune response in an individual or subject against one ormore tumor antigens.

As used herein, a cancer antigen is broadly defined as an antigenspecifically expressed by a tumour or cancer cell. A cancer antigenwhich is present on the surface of cancer cells in an individual butwhich is not present on the surface of normal somatic cells of theindividual i.e. the antigen is exposed to the immune system in cancercells but not in normal somatic cells. The antigen may be expressed atthe cell surface of the tumour cell where it is recognized by componentsof the humoral immune system such as B lymphocytes (B cells).Intracellular tumour antigens are processed into shorter peptidefragments which form complexes with major histocompatibility complex(MHC) molecules and are presented on the cell surface of cancer cells,where they are recognized by the T cell receptors (TCR's) of Tlymphocytes (T cells). Preferably, the cancer antigen is one which isnot expressed by normal cells, or at least not expressed to the samelevel as in tumour cells. An immunotherapeutic agent, such as a cancervaccine maybe comprised of one or more epitopes or antigenicdeterminants, e.g. peptide epitopes or antigenic determinants from atumor or cancer antigen, such that the immune response generated by thecancer vaccine is reactive against the antigen.

A cancer vaccine may enhance the presentation of one or more cancerantigens to both antigen presenting cells (e.g., macrophages anddendritic cells) and/or to other immune cells such as T cells, B cells,and NK cells. In some examples, preparations and/or formulations ofcancer vaccines may be used together with one or more adjuvants that arewell known in the art, to induce an immune response or to increase animmune response.

Cancer antigens may include, for example, cancer-testis antigens encodedby cancer-germ line genes. Cancer-testis (CT) antigens constitute aunique group of genes which are predominantly expressed in human germline cells such as placenta and testis but become reactivated in variousmalignancies (Simpson et al., Nature Rev (2005) 5, 615-625). Most ofthese genes are located as multigene families on the X-chromosome andare also referred to as CT-X antigens (Simpson et al., Nature Rev (2005)5, 615-625). Analogies have been drawn between their expression patternduring germ maturation and neoplastic transformation, thus suggestingtheir involvement in several steps of tumorigenesis (Simpson et al.,Nature Rev (2005) 5, 615-625). The CT-X antigens are broadly expressedin a wide variety of cancer types including for example bladder cancer,lung cancer, ovarian cancer, breast cancer, prostate cancer, Braincancer, glioma, glioblastoma, hepatocellular carcinoma and melanoma.Moreover, their expression pattern is closely associated with advanceddisease and poor outcome and might thus be of diagnostic and/orprognostic relevance (Gure et al., Clin Cancer Res (2005) 11,8055-8062;Velazquez et al., Cancer Immun (2007) 7, 11; Andrade et al., CancerImmun (2008) 8, 2; Tinguely et al., Cancer Science (2008); Napoletano etal., Am J of Obstet Gyn (2008) 198, 99 e91-97. Due to their highlyrestricted expression in malignant tissues, their tumour associatedpeptide epitopes provide promising targets for anticancer immunotherapy(Scanlan et al., Immunol Rev (2002) 188, 22-32). Indeed, clinical trialsevaluating the role of CT antigens, namely MAGE-A3, Prame and NY-ESO-I,as targets for specific immunotherapy have already been initiated in anumber of different malignancies (Bender et al., Cancer Immunol (2007)7, 16; Atanackovic et al., PNAS (2008) 105, 1650-1655; Jager et al.,PNAS (2006) 103, 14453-14458; van Baren et al., J Clin Oncol (2005) 23,9008-9021; Valmori et al., PNAS (2007) 104, 8947-8952; Odunsi et al.,PNAS (2007) 104, 12837-12842; Davis et al., PNAS (2004) 101, 10697-10702(9-15). Tumor antigens which may be comprised of the full-lengthpolypeptide sequence of the tumor antigen or an immunogenic fragment, orepitope derived from the full-length polypeptide sequence of the tumorantigen. Tumor antigens include the corresponding nucleotide sequenceencoding for the full-length polypeptide, immunogenic fragment, orepitope derived from the full-length polypeptide sequence of the tumorantigen.

A fragment of a cancer antigen is a contiguous stretch of amino acidresidues from the sequence of the antigen which is shorter than the fulllength antigen (i.e. it consists of fewer amino acid residues). Forexample, a fragment may comprise less than 500, less than 400, less than300, less than 200, less than 100 amino acids, or less than 50 aminoacids. A fragment will generally consist of at least 5 amino acids, forexample, at least 10 amino acids, at least 15 amino acids, at least 20amino acids, at least 25 amino acids, at least 30 amino acids or atleast 35 amino acids. Fragments of cancer antigens may includeimmunogenic regions or epitopes that bind to MHC class I or class IImolecules and are recognized by TCR's of T lymphocytes. Many suchepitopes of cancer antigens are known in the artwww.cancerimmunity.org/peptidedatabase/Tcellepitopes.

The cancer antigen can be a tumor associated peptide, or protein thatinduces or enhances immune response and is derived from tumor associatedgenes and encoded proteins including, for example, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C₁, MAGE-C₂, NY-ESO-1, LAGE-1,SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7,alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11,hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9,pml-RAR.alpha. fusion protein, PTPRK, K-ras, N-ras, Triosephosphateisomeras, GnTV, Herv-K-mel, Lage-1, Mage-C₂, NA-88, /Lage-2, SP17, andTRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3,p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu,BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens,EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4,MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H₁, PSA, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, .beta.-Catenin, CDK4, Mum-1, p16,TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72,.alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, and TPS. For example,antigenic peptides characteristic of tumors include those listed inInternational Patent Application Publication No. WO 20000/020581 andU.S. Patent Application Publication No. 2010/0284965, which are eachincorporated herein by reference. In some exemplary embodiments, theantigen is a tumor antigen selected from the group consisting of MUC1,MAGE. BAGE, RAGE, CAGE, SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase,melan-A, and mixtures thereof. In some variations, the cancer antigen isa mammalian protein. In some variations, the cancer antigen is a humanprotein. In some variations, the full-length protein is employed as theantigen. In some variations, peptides comprising an antigenic fragmentof these proteins is used as the tumor antigen.

Other suitable antigens include cancer antigens in the followingclasses: cancer testis antigens (e.g., HOM-MEL-40), differentiationantigens (e.g., HOM-MEL-55), overexpressed gene products (HOM-MD-21),mutated gene products (NY-COL-2), splice variants (HOM-MD-397), geneamplification products (HOM-NSCLC-11) and cancer related autoantigens(HOM-MEL-2.4) as reviewed in Cancer Vaccines and Immunotherapy (2000)Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge.Further examples include, MART-1 (Melanoma Antigen Recognized byT-cells-1) MAGE-A (MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8,MAGE-A10, MAGE-A12), MAGE B (MAGE-B1-MAGE-B24), MAGE-C(MAGE-C₁/CT7,CT10), GAGE (GAGE-1, GAGE-8, PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE(LAGE-1a(1S), -1b(1L), NY-ESO-1), SSX (SSX1-SSX-5), BAGE, SCP-1, PRAME(MAPE), SART-1, SART-3, CTp11, TSP50, CT9/BRDT, gp100, MART-1, TRP-1,TRP-2, MELAN-A/MART-1, Carcinoembryonic antigen (CEA), prostate-specificantigen (PSA), MUCIN (MUC-1) and Tyrosinase. TAAs are reviewed in CancerImmunology (2001) Kluwer Academic Publishers, The Netherlands.Additional cancer associated antigens include Her 2, survivin and TERT.

The term “antigen” refers to protein or peptide to be introduced into asubject. As described herein, an antigen may be provided throughdelivering a peptide or protein or through delivering a nucleic acidencoding a peptide or protein.

By “antigen” in the context of the present disclosure it is also meantto incorporate an antigenic peptide derived from an antigen. Inparticular, “cancer associated antigen” is intended to encompass apeptide derived from a cancer associated antigen.

An antigen such as a cancer associated antigen can be provided for useas a medicament in a number of different ways. It can be administered aspart of a vector.

Any suitable vector may be used to introduce a polynucleotide thatencodes a polypeptide of the invention encoding one of the tumor antigenproteins into the host. Exemplary vectors that have been described inthe literature include replication deficient retroviral vectors,including but not limited to lentivirus vectors [Kim et al., J. Virol.,72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October,1998, pp. 43 46.]; adenoassociated viral (AAV) vectors [U.S. Pat. Nos.5,474,935; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390;5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al., J. Invest.Med., 45: 87 98 (1997)]; adenoviral (AV) vectors [See, e.g., U.S. Pat.Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362;Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581 2584 (1992);Stratford Perricadet et al., J. Clin. Invest., 90: 626 630 (1992); andRosenfeld et al., Cell, 68: 143 155 (1992)]; an adenoviraladenoassociated viral chimeric (see for example, U.S. Pat. No.5,856,152) or a vaccinia viral or a herpesviral (see for example, U.S.Pat. Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688;Lipofectin mediated gene transfer (BRL); liposomal vectors [See, e.g.,U.S. Pat. No. 5,631,237 (Liposomes comprising Sendai virus proteins)];and combinations thereof.

Suitable cancer vaccines are known in the art and may be produced by anyconvenient technique.

For example, a cancer vaccine may be generated wholly or partly bychemical synthesis. For example, a peptide-based vaccine or immunogenmay be synthesised using liquid or solid-phase synthesis methods; insolution; or by any combination of solid-phase, liquid phase andsolution chemistry, e.g. by first completing the respective peptideportion and then, if desired and appropriate, after removal of anyprotecting groups being present, by introduction of the residue X byreaction of the respective carbonic or sulfonic acid or a reactivederivative thereof. Chemical synthesis of peptides is well-known in theart (J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2ndedition, Pierce Chemical Company, Rockford, Ill. (1984); M. Bodanzskyand A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag,New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. OxfordUniversity Press, Oxford 1991; in Applied Biosystems 430A Users Manual,ABI Inc., Foster City, Calif.; G. A. Grant, (Ed.) Synthetic Peptides, AUser's Guide. W. H. Freeman & Co., New York 1992, E. Atherton and R. C.Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press1989 and in G. B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methodsin Enzymology Vol. 289). Academic Press, New York and London 1997).

Alternatively, peptide-based cancer vaccines may be generated wholly orpartly by recombinant techniques. For example, a nucleic acid encoding acancer antigen may be expressed in a host cell and the expressed antigenisolated and/or purified from the cell culture. For example, antigen maybe expressed in E. coli either in soluble form or in inclusion bodies,which may be solubilized and refolded. After expression, the antigen maybe isolated and/or purified. Cancer antigen may be analyzed by standardtechniques, such as mass spectrometry and western blot analysis.

The use of tumor antigens to generate immune responses iswell-established in the art (see for example; Kakimi K, et al. Int JCancer. 2011 Feb. 3; Kawada J, Int J Cancer. 2011 Mar. 16; Gnjatic S, etal. Clin Cancer Res. 2009 Mar. 15; 15(6):2130-9; Yuan J, et al. ProcNatl Acad Sci USA. 2008 Dec. 23; 105(51):20410-5; Sharma P, et al. JImmunother. 2008 November-December; 31(9):849-57; Wada H, et al. Int JCancer. 2008 Nov. 15; 123(10):2362-9; Diefenbach C S, et al. Clin CancerRes. 2008 May 1; 14(9):2740-8; Bender A, et al. Cancer Immun. 2007 Oct.19; 7:16; Odunsi K, et al. Proc Natl Acad Sci USA. 2007 Jul. 31;104(31):12837-42; Valmori D, et al. Proc Natl Acad Sci USA. 2007 May 22;104(21):8947-52; Uenaka A, et al. Cancer Immun. 2007 Apr. 19; 7:9;Kawabata R, et al. Int J Cancer. 2007 May 15; 120(10):2178-84; Jäger E,et al. Proc Natl Acad Sci USA. 2006 Sep. 26; 103(39):14453-8; Davis I DProc Natl Acad Sci USA. 2005 Jul. 5; 102(27):9734; Chen Q, Proc NatlAcad Sci USA. 2004 Jun. 22; 101(25):9363-8; Jäger E, Proc Natl Acad SciUSA. 2000 Oct. 24; 97(22):12198-203; Carrasco J, et al. J Immunol. 2008Mar. 1; 180(5):3585-93; van Baren N, et al. J Clin Oncol. 2005 Dec. 10;23(35):9008-21; Kruit W H, et al. Int J Cancer. 2005 Nov. 20;117(4):596-604; Marchand M, et al. Eur J Cancer. 2003 January;39(1):70-7; Marchand M et al. Int J Cancer. 1999 Jan. 18; 80(2):219-30;Atanackovic D, et al. Proc Natl Acad Sci USA. 2008 Feb. 5;105(5):1650-5).

Typically, an immunotherapeutic agent, such as a cancer vaccine, isadministered to the individual whose cancer expresses the said antigen.Cancer cells from the individual may be analyzed to identify the cancerantigen and patients are then identified for administration of theappropriate immunotherapeutic agent or cancer vaccine. For example, amethod as described herein may comprise the step of identifying a cancerantigen which is displayed by one or more cancer cells in a sampleobtained from the individual.

A biological sample may be obtained from the subject such as a biopsy,blood or bone marrow sample and tested for the presents of cancer cellswhich may be identified as displaying the cancer antigen using anystandard techniques including but not limited to immunologicaltechniques, such as immunocytochemistry and immunohistochemistry may beemployed. Additional techniques include immunological analysis such asserologically determining an autologous immune response to said cancerantigen, see WO2001/007917. Analysis of gene expression can be performedusing methods known in the art such as polymerase chain reaction ormicroarray analysis.

Combination Therapy Compositions:

A pharmaceutical composition may comprise, in addition to (1) thecompound as disclosed herein, (2) an immunotherapeutic agent such as acancer vaccine, (3) an adjuvant, and (4) a pharmaceutically acceptableexcipient, carrier, buffer, stabilizer or other materials well known tothose skilled in the art. Suitable materials will be sterile and pyrogenfree, with a suitable isotonicity and stability. Examples ofpharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials include sterile saline (e.g. 0.9% NaCl), water,dextrose, glycerol, ethanol or the like or combinations thereof. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active compound. The precise nature of the carrier or othermaterial will depend on the route of administration, which may be bybolus, infusion, injection or any other suitable route, as discussedbelow. The composition may further contain auxiliary substances such aswetting agents, emulsifying agents, pH buffering agents or the like.

An adjuvant is a substance incorporated into or administered withantigen which potentiates the immune response. Adjuvants may enhance theimmunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella Minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKlineBeecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., MoI Cells (1997) 7:178-186); ISCOMATRIX adjuvant,a cage-like structure composed of saponin, phospholipid, and cholesterol(see, e.g., Maraskovsky et al., Clin. Cancer Res. (2004) 10:2879-2890);incomplete Freund's adjuvant; complete Freund's adjuvant; montanide;alum; CpG oligonucleotides (see e.g. Kreig et al., Nature 374:546-9,1995) and other immunostimulatory oligonucleotides including poly-IC andpoly-ICLC (Hiltonol®); and various water-in-oil emulsions prepared frombiodegradable oils such as squalene and/or tocopherol.

The cancer vaccine may be administered in conjunction with an adjuvant.Suitable adjuvants are well known in the art and include aluminum salts,such as alum (aluminium potassium sulphate dodecahydrate), aluminumhydroxide and aluminum phosphate and organic compounds, such assqualene.

In addition to a cancer antigen, an immunotherapeutic, immunogenic orvaccine formulation may comprise an adjuvant. For example, a formulationmay comprise 1-500 μg, preferably 1-50 μg, of cancer antigen and 0.5 to20 mg, preferably 1-10 mg, of adjuvant in a pharmaceutically acceptablecarrier or diluent as mentioned above.

A vaccine formulation may comprise a Toll-like Receptor (TLR) ligand.Suitable TLR ligands include polyinosinicpolycytidylic acid (poly I:C),lipopolysaccharide (LPS), CpG oligodeoxynucleotide, poly LC, poly ICLC,MPL (Corixa Corp) and imidazoquinolines, such as imiquimod and R848. Theuse of TLR ligands to modulate immune responses is well-known in the art(see for example, Weiner et al (1997) PNAS USA 94 10833-10837; Vabulaset al J. Immunol. (2000) 164 2372-2378; Gunzer et al (2005) Blood 1062424-2432).

Formulations of immunotherapeutic agents, such as cancer vaccines, arewell-known in the art and include MAGE-A3 ASCI, NY-ESO-1 ASCI and PRAMEASCI (GSK Bio); Provenge (Dendreon), Abogovomab (Meranini), M-Vax(Avax), Allovectin-7 (Vial) for metastatic melanoma, GSK1572932A (GSKBio) Belagenpumatucel-L (Novarex) BMP-25 (Merck Serono), BiovaxID(Biovest/Accentia), MDX-1379 (Medarex/BMS), Ipilimumab (BMS) Trovax(Oxford Biomedical) Oncophage (Antigenics) and PR1 leukemia peptide (TheVaccine company).

The invention will be more fully understood by reference to thefollowing examples which detail exemplary embodiments of the invention.They should not, however, be construed as limiting the scope of theinvention. All citations throughout the disclosure are hereby expresslyincorporated by reference.

EXAMPLES Synthesis of Compounds

6-iodo-((2R,3S,4R)-benzyloxy)-methyl-α-D-galactose (46)

A solution of glycoside 45/49/52 (206 mg, 0.44 mmol) and PPh₃ (139 mg,0.53 mmol) in toluene (5 mL) was heater under reflux with for 10 min.The reaction mixture was cooled to 80° C., and then imidazole (89 mg,1.32 mmol) and 12 (142 mg, 0.57 mmol) were added. The mixture was heatedunder reflux for 20 min before being concentrated under reducedpressure. The residue was dissolved in EtOAc(50 mL) and washed withNa₂S₂O₃ solution (20 mL) and H₂O (20 mL). The organic layer was thendried over Na₂SO₄, filtered and the filtrate concentrated under reducedpressure. The crude product was purified by column chromatography (8%EtOAc in hexanes) to give iodide 46 as a colourless oil (183 mg, 72%).

(2R,3S,4S)-2,3,4-tris(benzyloxy)hex-5-enal (47)

Zinc dust was preactivated by stirring in HCl (50 mL of 1.0 M solution)at rt for 15 mins, before being filtered and washed sequentially withH₂O (30 mL), acetone (30 mL) and Et₂O (30 mL). The zinc was then driedunder high vacuum with a heatgun. The activated zinc (0.706 mg, 10.8mmol) was added to a solution of glycoside 46 (620 mg, 1.08 mmol) andTMSCl (0.137 mL, 1.08 mmol) in THF (20 mL) and the reaction mixturesonicated at 40° C. After 5 h Et₂O (50 mL) and H₂O (50 mL) were added tothe suspension, which was then filtered through Celite. The layers wereseparated and the aqueous layer was extracted with Et₂O (3×25 mL). Thecombined organic layers were washed with H₂O (2×15 mL) and brine (15mL), then dried over Na₂SO₄, filtered and the filtrate concentratedunder reduced pressure. The crude product was purified by columnchromatography (8% EtOAc in hexanes) to give aldehyde 47 as a colourlessoil.

6-((tert-butyldiphenylsilyl)oxy)-methyl-α-D-galactose (50)

Imidazole (0.77 g, 11.30 mmol), and TBDPSCl (1.74 mL, 6.70 mmol) wereadded sequentially to a solution of methyl □-D-galactopyranoside (1.00g, 5.15 mmol) in DMF (5 mL). After 24 h the reaction mixture was dilutedwith Et₂O (30 mL), washed with H₂O (20 mL) and NH₄Cl solution (20 mL).The organic layer was dried over Na₂SO₄, filtered and the filtrateconcentrated under reduced pressure. The crude product was purified bycolumn chromatography (30% hexanes in EtOAc) to give silyl ether 50 as acolourless oil (2.10 g, 94%).

2,3,4-benzyloxy-6-((tert-butyldiphenylsilyl)oxy)-methyl-α-o-galactose(51)

NaH (60% wt in mineral oil, 0.61 g, 15.5 mmol) was added to a solutionof glycoside 50 (1.20 g, 2.78 mmol) in DMF (10 mL) at 0° C. The reactionmixture was stirred for 20 min, then BnBr (1.33 mL, 11.1 mmol) was addedat 0° C. After stirring overnight at rt the reaction was quenched withthe slow addition of MeOH, and then diluted with EtOAc (30 mL). Theorganic layer was washed with H₂O (20 mL), dried over Na₂SO₄, filteredand the filtrate concentrated under reduced pressure. The crude productwas purified by column chromatography (10% EtOAc in hexanes) to givetribenzyl ether 51 as a colourless oil.

2,3,4-tribenzyloxy-methyl-α-D-galactose (52)

TBAF (1M solution in THF, 1.27 mL, 1.27 mmol) was added to a solution ofglycoside 51 (460 mg, 0.63 mmol) in THF (5 mL). The reaction mixture wasstirred overnight before being quenched with H₂O (15 mL). The resultinglayers were separated and the aqueous layer was extracted with EtOAc(3×15 mL). The organic layers were combined and washed with brine (15mL), dried over Na₂SO₄, filtered and the filtrate concentrated underreduced pressure to provide the crude product, which was used directlyin the next reaction without further purification. Rf=0.19 (40% EtOAc inhexanes)

(5S,6S,7S)-5,6,7-tris(benzyloxy)nona-1,8-dien-4-ol (53)

Allyl magnesium bromide (1.0 M in Et₂O, 1.44 mL, 1.44 mmol) was addeddropwise over 5 min to a solution of aldehyde 47 (200 mg, 0.48 mmol) inTHF (10 mL) at −78° C. The reaction mixture was left stirring at thistemperature for 4 h before being quenched with NH₄Cl solution (30 mL).The resulting layers were separated and the aqueous layer was extractedwith EtOAc (3×25 mL). The combined organic layers were washed with H₂O(20 mL) and brine (20 mL), dried over Na₂SO₄, filtered and the filtrateconcentrated under reduced pressure. The crude product was purified bycolumn chromatography (0-4% EtOAc in hexanes, gradient) to give product53 as a mixture of two diasteroisomers.

(4R,5S,6S,7S)-5,6,7-tris(benzyloxy)cyclohept-2-enol (54a) and(4S,5S,6S,7S)-5,6,7-tris(benzyloxy)cyclohept-2-enol (54b)

A solution of diene 53 (270 mg, 0.59 mmol) in CH₂Cl₂ (60 mL) wasdegassed according to the general procedure. Grubbs 2^(nd) generation Rumetathesis catalyst (8 mg, 0.009 mmol) was added and the solution washeated under reflux. After 2 h the solution was concentrated underreduced pressure and the crude product purified by column chromatography(20% EtOAc in hexanes) to give products 54a and 54b.

(2R,3R,4R)-2-amino-1-((tert-butyldiphenylsilyl)oxy)octadecane-3,4-diol(64)

TBDPSCl (2.46 mL, 9.45 mmol) was added to a solution of phytosphingsine(2.0 g, 6.3 mmol) in pyridine. After stirring overnight the reaction wasquenched with MeOH (5 mL), then the solvent was removed under reducedpressure. The crude product was purified by column chromatography (50%EtOAc in hexanes, EtOAc, 0%-7% MeOH in EtOAc) to provide sphingosine 64as a colourless oil.

2R—N-benzyl-1-((tert-butyldiphenylsilyl)oxy)-(3R,4R—O-isopropylidene)octadecane (65)

Benzaldehyde (86 μL, 0.85 mmol) was added to a stirred suspension ofamine 66 (420 mg, 0.71 mmol) and NaBH(OAc)₃ (377 mg, 1.78 mmol) in THF(5 mL). After stirring overnight the reaction mixture was diluted withEt₂O (20 mL) and NaHCO₃ solution (20 mL). The resulting layers wereseparated and the aqueous layer was extracted with Et₂O (3×20 mL). Theorganic layers were combined and washed with brine (20 mL), then driedover Na₂SO₄, filtered and the filtrate concentrated under reducedpressure. The crude product was purified by column chromatography (0-2%EtOAc in hexanes, gradient) to give amide 65 as a colourless oil.

2R-amino-1-((tert-butyldiphenylsilyl)oxy)-(3R,4R—O-isopropylidene)octadecane(66)

Concentrated H₂SO₄ (4 drops) was added to a solution of sphingosine 64(450 mg, 0.81 mmol) in dry acetone (10 mL) at 0° C. and stirred for 5 h.The reaction mixture was quenched with NaHCO₃ solution (20 mL), thenconcentrated under reduced pressure. The mixture was then extracted withEtOAc (3×20 mL) and the combined organic layers were washed with brine(10 mL), then dried over Na₂SO₄, filtered and the filtrate concentratedunder reduced pressure. The crude product was purified by columnchromatography (20% EtOAc in hexanes) to provide acetonide 67 as acolourless oil.

2R—N-benzyl-(3R,4R—O-isopropylidene)octadecan-1-ol (68)

TBAF (1M solution in THF, 1.27 mL, 1.27 mmol) was added to a solution ofacetonide 66 (440 mg, 0.64 mmol) in THF (20 mL). The reaction mixturewas stirred overnight before being quenched with H₂O (15 mL). Theresulting layers were separated and the aqueous layer was extracted withEtOAc (3×15 mL). The organic layers were combined and washed with brine(15 mL), dried over Na₂SO₄, filtered and the filtrate concentrated underreduced pressure. The crude product was purified by columnchromatography (25% EtOAc in hexanes) to provide alcohol 68 as acolourless oil.

(R)-3-benzyl-4-((4R,5R)-2,2-dimethyl-5-tetradecyl-1,3-dioxolan-4-yl)-1,2,3-oxathiazolidine2,2-dioxide (70)

A solution of amide 68 (280 mg, 0.63 mmol) in CH₂Cl₂ (5 mL) was addeddropwise over 30 min to a −50° C. solution of SOC12 (50 μL, 0.69 mmol),imidazole (172 mg, 2.52 mmol) and NEt₃ (194 μL, 1.39 mmol) in CH₂Cl₂ (6mL). The reaction mixture was warmed up to 0° C. and stirred for 21 h,before adding H₂O (10 mL). The organic layer was isolated and washedwith brine (5 mL), dried over Na₂SO₄, filtered and the filtrateconcentrated under reduced pressure to give the crude sulfamidite, whichwas used immediately. Rf=0.88 (30% EtOAc in hexanes)

NaIO₄ (148 mg, 0.69 mmol), RuCl₃ (14 mg, 0.064 mmol) and H₂O (5 mL) wereadded sequentially to a solution of crude sulfamidite in MeCN (5 mL) at0° C. After 2.5 h the reaction mixture was diluted with H₂O (50 mL) andEt₂O (50 mL). The resulting layers were separated and the aqueous layerwas extracted with Et₂O (3×35 mL). The organic layers were combined andwashed with H₂O (30 mL), brine (20 mL), then dried over Na₂SO₄, filteredand the filtrate concentrated under reduced pressure. The crude productwas purified by column chromatography (10% EtOAc in hexanes) to givesulfamidate 70 as a colourless oil.

(R)—N-benzyl-1-((4R,5S)-2,2-dimethyl-5-tetradecyl-1,3-dioxolan-4-yl)-2-(((1R,5S,6S,7S)-5,6,7-tris(benzyloxy)cyclohept-3-en-1-yl)oxy)ethanamine(71)

NaH (40% wt in mineral oil, 10 mg, 0.24 mmol) was added to a solution ofalcohol 54b (34 mg, 0.079 mmol) in DMF (0.25 mL) and THF (0.05 mL) at 0°C. After stirring for 1 h a solution of sulfamidate 70 (40 mg, 0.079mmol) in THF (0.2 mL) was added at 0° C. After stirring overnight at 40°C. the reaction mixture was concentrated and the residue was dissolvedin Et₂-O (10 mL). A 20% aq. H₂SO₄ solution (10 mL) was added at 0° C.and the reaction mixture was stirred for 20 min before being neutralizedwith K₂CO₃ (1 g). After 40 min Et₂O (20 mL) and H₂O (20 mL) was added.The resulting layers were separated and the aqueous layer was extractedwith Et₂O (3×35 mL). The organic layers were combined and washed withH₂O (30 mL), NaHCO₃ solution (20 mL) and brine (20 mL), then dried overNa₂SO₄, filtered and the filtrate concentrated under reduced pressure.The crude product was purified by column chromatography (10% EtOAc inhexanes) to give ether 71 as a colourless oil.

(1S,2S,3R,4R)-4-(((2R,3R,4S)-2-amino-3,4-dihydroxyoctadecyl)oxy)cycloheptane-1,2,3-triol (72)

A 1M solution of HCl (150 μL, 0.15 mmol) and Pd—C(10% wt, 32 mg, 0.03mmol) were added to a solution of ether 71 (130 mg, 0.15 mmol) andcyclohexene (2 mL) in MeOH (10 mL) and heated under reflux. Afterstirring overnight the reaction mixture was cooled to rt and dilutedwith a 5:1 solution of CHCl₃: MeOH, before being filtered thought a bedof celite. The filtrate was concentrated under reduced pressure toprovide the crude amine 72, which was used directly in the next step.

N-((2R,3R,4S)-3,4-dihydroxy-1-(((1S,2R,3S,4S)-2,3,4-trihydroxycycloheptyl)oxy)octadecan-2-yl)hexacosanamide (73)

(COCl)₂ (2 mL) was added to hexacosanoic acid (139 mg, 0.39 mmol) andheated at 70° C. for 2 h, after which time the solution was cooled tort, and the (COCl)₂ removed under a stream of dry argon. The residualvolatiles were removed under reduced pressure. The resulting crude acylchloride was dissolved in THF (0.5 mL) and added with vigorous stirringto a solution of amine 72 (81 mg, 0.18 mmol) in THF/NaOAc_((aq)) (8M)(1:1, 2 mL). Vigorous stirring was maintained for 2 h, after which timethe reaction mixture was left to stand and the layers were separated.The aqueous layer was extracted with THF (3×2.0 mL) and the organiclayers were combined and concentrated under reduced pressure. The crudeproduct was purified by column chromatography (10% MeOH in CHCl₃) togive amide 73 as a white solid.

(1S,1′S)-1,1′-((4S,5S)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)bis(prop-2-en-1-ol)(75)

A solution of (2R,3R)-2,3-O-isopropylidene tartrate (1.5 g, 6.9 mmol) intoluene (25 mL) was degassed according to the general procedure. DIBALH(1.0 M in toluene, 14.4 mL, 14.4 mmol) was added dropwise over 10 minsto the solution at −78° C. which was left to stir for 2.5 h at thattemperature. After 2.5 h vinyl magnesium bromide (1.0 M in THF, 20.6 mL,20.6 mmol) was added and the reaction mixture left to stir for 2 h at−78° C., before being allowed to warm up to rt slowly. The reaction wascarefully quenched with NH₄Cl solution (50 mL) and the resulting layerswere separated. The aqueous layer was extracted with EtOAc (3×35 mL).The organic layers were combined and washed with H₂O (20 mL) and brine(20 mL), then dried over Na₂SO₄, filtered and the filtrate concentratedunder reduced pressure. The crude product was purified by columnchromatography (25% EtOAc in hexanes) to give diene 75 as the majorproduct in a mixture of diastereoisomers with a ratio of 3:1.

3S,3'S—O-isopropylidene-cyclohex-1-en-2S,2'S-diol (76)

A solution of diene 75 (217 mg, 1.01 mmol) in CH₂Cl₂ (230 mL) wasdegassed according to the general procedure. Grubbs 2^(nd) generation Rumetathesis catalyst (12 mg, 0.015 mmol) was added and the solution washeated under reflux. After 2 h the solution was concentrated underreduced pressure and the crude product purified by column chromatography(5% MeOH in CHCl₃) to give diol 76.

4S-(tert-butyldimethylsily)oxy)-5S,6S—O-isopropylidene-cyclohex-2-enol(77)

Imidazole (275 mg, 4.04 mmol) and TBDMSCl (486 mg, 3.23 mmol) were addedsequentially to a solution of diol 76 (500 mg, 2.69 mmol) in DMF (5 mL).After stirring overnight the reaction mixture was diluted with Et₂O (30mL), washed with H₂O (15 mL) and NH₄Cl solution (15 mL). The organiclayer was dried over Na₂SO₄, filtered and the filtrate concentratedunder reduced pressure. The crude product was purified by columnchromatography (20% EtOAc in hexanes) to give alcohol 77 as a colourlessoil.

(2′R)—N-benzyl-1-((1S,2S,3S,4S)-4-((tert-butyldimethylsilyl)oxy)-2,3-O-isopropylidene-cyclohex-5-ene)-3′R,4'S—O-isopropylidene-octadecane(78)

NaH (40% wt in mineral oil, 78 mg, 1.95 mmol) was added to a solution ofalcohol 77 (195 mg, 0.65 mmol) in DMF (2 mL) and THF (1 mL) at 0° C.After stirring for 1 h a solution of sulfamidate 70 (397 mg, 0.78 mmol)in THF (1 mL) was added at 0° C. After stirring overnight at 40° C. thereaction mixture was concentrated and the residue was dissolved in Et₂O(10 mL). A 20% aq. H₂SO₄ solution (10 mL) was added at 0° C. and thereaction mixture was stirred for 20 min before being neutralized withK₂CO₃ (1 g). After 40 min Et₂₀ (20 mL) and H₂O (20 mL) was added. Theresulting layers were separated and the aqueous layer was extracted withEt₂₀ (3×35 mL). The organic layers were combined and washed with H₂O (30mL), NaHCO₃ solution (20 mL) and brine (20 mL), then dried over Na₂SO₄,filtered and the filtrate concentrated under reduced pressure. The crudeproduct was purified by column chromatography (10% EtOAc in hexanes) togive ether 78 as a colourless oil.

(2R,3R,4S)-2-amino-1-((1S,2S,3S,4S)-4-((tert-butyldimethylsilyl)oxy)-2,3-O-isopropylidene-cyclohexane)octadecane-3,4-diol(79)

A 1M solution of HCl (71 μL, 0.071 mmol) and Pd—C(10% wt, 15 mg, 0.014mmol) were added to a solution of ether 78 (50 mg, 0.071 mmol) andcyclohexene (1 mL) in MeOH (5 mL) and heated under reflux. Afterstirring overnight the reaction mixture was cooled to rt and dilutedwith a 5:1 solution of CHCl₃: MeOH, before being filtered thought a bedof celite. The filtrate was concentrated under reduced pressure toprovide the crude amine 79, which was used directly in the next step.

N-((2R,3R,4S)-3,4-dihydroxy-1-(((1S,2R,3S,4S)-2,3,4-trihydroxycyclohexyl)oxy)octadecan-2-yl)hexacosanamide (81)

Neat TFA (2 mL) was added to ether 79 (assuming 100% conversion, 0.071mmol) for 15 min before removal of the TFA under reduced pressure. Thisprocedure was repeated if necessary to provide the crude amine 80, whichwas used directly in the next reaction. Rf=0.33 (30% MeOH in CHC13)

(COCl)₂ (2 mL) was added to hexacosanoic acid (21 mg, 0.054 mmol) andheated at 70° C. for 2 h, after which time the solution was cooled tort, and the (COCl)₂ removed under a stream of dry argon. The residualvolatiles were removed under reduced pressure. The resulting crude acylchloride was dissolved in THF (0.5 mL) and added with vigorous stirringto a solution of amine 80 (20 mg, 0.045 mmol) in THF/NaOAc_((aq)) (8M)(1:1, 2 mL). Vigorous stirring was maintained for 2 h, after which timethe reaction mixture was left to stand and the layers were separated.The aqueous layer was extracted with THF (3×2.0 mL) and the organiclayers were combined and concentrated under reduced pressure. The crudeproduct was purified by column chromatography (10% MeOH in CHCl₃) togive amide 81 as a white solid.

(1S,1′S)-1,1′-((4S,5S)-2,2-dimethyl-1,3-dioxolane-4,5-diyl)bis(but-3-en-1-ol)(89)

A solution of (2R,3R)-2,3-O-isopropylidene tartrate (1.88 g, 8.6 mmol)in toluene (25 mL) was degassed. DIBALH (1.0 M in toluene, 18.1 mL, 18.1mmol) was added dropwise over 10 mins to the solution at −78° C. whichwas left to stir for 2.5 h at that temperature. After 2.5 h allylmagnesium bromide (1.0 M in THF, 25.9 mL, 25.9 mmol) was added and thereaction mixture left to stir for 2 h at −78° C., before being allowedto warm up to rt slowly. The reaction was carefully quenched with NH₄Clsolution (50 mL) and the resulting layers were separated. The aqueouslayer was extracted with EtOAc (3×35 mL). The organic layers werecombined and washed with H₂O (20 mL) and brine (20 mL), then dried overNa₂SO₄, filtered and the filtrate concentrated under reduced pressure.The crude product was purified by column chromatography (25% EtOAc inhexanes) to give diene 89.

4S,4'S—O-isopropylidene-cyclooct-1-en-4,4′-diol (90)

A solution of diene 89 (400 mg, 1.65 mmol) in CH₂Cl₂ (750 mL) wasdegassed. Grubbs 2^(nd) generation Ru metathesis catalyst (21 mg, 0.025mmol) was added and the solution was heated under reflux. After 2 h thesolution was concentrated under reduced pressure and the crude productpurified by column chromatography (40% EtOAc in hexanes) to give diol90.

6-(tert-butyldimethylsilyl)oxy)-7S,8S—O-isopropylidene-cyclooct-3-en-1-ol(91)

Imidazole (140 mg, 2.1 mmol) and TBDMSCl (187 mg, 1.2 mmol) were addedsequentially to a solution of diol 90 (220 mg, 1.0 mmol) in DMF (5 mL).After stirring overnight the reaction mixture was diluted with Et₂O (30mL), washed with H₂O (15 mL) and NH₄Cl solution (15 mL). The organiclayer was dried over Na₂SO₄, filtered and the filtrate concentratedunder reduced pressure. The crude product was purified by columnchromatography (20% EtOAc in hexanes) to give alcohol 91 as a colourlessoil.

Biology—Linker Analogues

The biological activity of each of the α-GalCer thioamide 6, α-GalCerurea 7, and α-GalCer carbamate 8 analogues and the ThrCer thioamide 9,ThrCer urea 10, and ThrCer caarbamate 11 analogues was investigatedalongside α-GalCer 1 and ThrCer 2. In a preliminary screen, all eightcompounds were tested for their ability to stimulate the iNKT cellhybridoma DN32, following pulsing of C1R-mCD1d cells with variousconcentrations of ligands. The concentration of IL-2 in the supernatantreleased after iNKT cell activation was measured using an enzyme-linkedimmunosorbent assay (ELISA). Encouragingly, these experimentsdemonstrated that both ThrCer-thioamide 9 and ThrCer-carbamate 11induced increased activation compared with ThrCer 2, whereas theThrCer-urea analogue 10 led to weak stimulation and only at highconcentrations (FIG. 1B). A similar hierarchy was observed for theα-GalCer analogues, although the differences, particularly at highconcentration, were less pronounced (FIG. 1A).

In another in vitro experiment to test functional activity, this timeusing a human model, human iNKT cells were co-cultured for 40 h withC1R-hCD1d cells that had been pulsed with 100 ng mL⁻¹ concentrations ofvehicle, α-GalCer 1, α-GalCer-thioamide 6, α-GalCer-urea 7, andα-GalCer-carbamate 8 (FIG. 2A) and, ThrCer 2, ThrCer-thioamide 9,ThrCer-urea 10, and ThrCer-carbamate 11 (FIG. 2B). In this assay, theability of the various ligands to activate iNKT cells was assessed bydetermining the levels of IFNγ production after 40 h by ELISA. Onceagain, all three ThrCer analogues were shown to stimulate human iNKTcells, albeit at lower levels than the αGalCer analogues, which is inaccord with the behaviour of the two parent compounds. In agreement withthe murine iNKT cell data (FIGS. 1A-1B), the weakest ligand at 100 ngmL⁻¹ was again ThrCer-urea 10; however in this assay, ThrCer-thioamide 9and ThrCer-carbamate 11 were now more comparable to ThrCer in theirbehaviour (FIG. 2B). All of the α-GalCer analogues stimulated human iNKTcells, with the urea analogue 7 proving to be the weakest activator atlow concentrations (FIG. 2A).

Since the two urea derivatives 7 and 10 displayed the weakest activityin in vitro experiments, further studies focused solely on the thioamideand carbamate derivatives of ThrCer and α-GalCer. These analogues werenext investigated in vivo, alongside the parent compounds andTh2-biasing molecule OCH9 (12, below), specifically to assess theirability to effect DC maturation as well as their cytokine responseprofile.

To this end, 1 μg lipid was injected intravenously (i.v.) into wildtypeC57 BL/6 or C57 BL/6 CD1d−/− (NKT cell-deficient) mice. After 2 h, themice were tail-bled and IL-4 levels in the serum measured by ELISA(FIGS. 3A-3B). At 18 h, the mice were sacrificed and blood serum levelsof IFNγ were measured by ELISA (FIGS. 3A-3B), and cells harvested fromthe spleen were used to determine the extent of DC maturation bymeasuring the expression of the co-stimulatory molecule, CD86 usingfluorescence-activated cell sorting (FACS) analysis (FIGS. 4A-4B).

The in vivo activation of iNKT cells with the α-GalCer and ThrCeranalogues was determined by analyzing the cytokine profile in bloodserum at 2 h and 18 h. Thus, α-GalCer analogues 6 and 8 showed a markeddecrease in the ability to stimulate iNKT cells to produce IL-4 at 2 hpost injection compared with α-GalCer, but both compounds were able tomaintain IFNγ production at 18 h, consistent with that of α-GalCer (FIG.3A). Differences in cytokine production were even more pronounced withthe weaker ThrCer agonists 9 and 11, both of which did not stimulateiNKT cells to produce IL-4 at all when assayed at 2 h, but were stillable to produce IFNγ at 18 h (FIG. 3B). No cytokine production wasdetected in CD1d−/− mice injected with the α-GalCer and ThrCeranalogues. Since the presentation of CD1d-lipid complex by DC to iNKTcells results in activation and the subsequent maturation of DC, we alsodetermined whether there was any difference in the ability of DC toupregulate the co-stimulatory molecule, CD86, following i.v. delivery ofα-GalCer and ThrCer analogues. Pleasingly, both sets of analoguesinduced DC maturation to a similar degree as the parent α-GalCer andThrCer compounds in wildtype mice but not in CD1d−/− mice (FIGS. 4A-4B).

The binding kinetics of these ThrCer and α-GalCer analogue compoundswere examined. To this end, bacterially-expressed hCD1d andβ-2-microglobulin (β 2 M) molecules were refolded with the thioamide,and carbamate analogues of both α-GalCer and ThrCer by oxidativerefolding chromatography, and then biotinylated. The urea analogues ofα-GalCer and ThrCer could not be refolded, and therefore, no surfaceplasmon resonance (SPR) data are available for these molecules. Solublehuman iNKT TCR was prepared. SPR experiments were used to measure theaffinity and kinetics of human iNKT cell TCRs for hCD1d loaded withα-GalCer, ThrCer, and their thioamide and carbamate analogues (FIGS.5A-5B). To this end, increasing concentrations of TCR were injected for5 seconds over the indicated complex immobilized on the BIAcore chipuntil the specific binding reached its plateau. K_(d) and B_(max) werecalculated by fitting the data using non-linear regression bindingkinetics model (FIGS. 5A-5B). Kinetic measurements for the k_(off) werecalculated using BIAevaluation software kit; k_(on) values werecalculated from the experimental k_(off) and K_(d).

TABLE experimental experimental calculated k_(on) lipid on CD1d K_(d) (μM) k_(off) (s⁻¹) (× 10³ M⁻¹ s⁻¹) ThrCer 2  4.57 ± 0.12  1.18 ± 0.0342.58 ± 0.10 ThrCer thioamide 9 36.06 ± 0.96  1.88 ± 0.045 5.20 ± 0.26ThrCer carbamate 11  4.60 ± 0.13  1.19 ± 0.072 2.59 ± 0.17 α-GalCer 1 2.19 ± 0.07 0.565 ± 0.008 2.58 ± 0.09 α-GalCer thioamide 6  4.20 ± 0.150.537 ± 0.011 1.28 ± 0.05 α-GalCer carbamate 8  1.72 ± 0.10 0.919 ±0.035 5.34 ± 0.37

The mechanisms by which glycolipid CD1d agonists are able to modulatethe cytokine response on iNKT cell activation are complex. The stabilityof the glycolipid-CD1d complex, and its TCR affinity have both beeninvoked to be important; however more recently the metabolic stabilityof the glycolipid, along with the cellular location of loading have alsobeen proposed to play a role. Indirect effects resulting from iNKT cellactivation also need to be considered; for example the Th2-biasingmolecule, OCH9 12, has been shown to reduce the level of CD40Lexpression by activated iNKT cells, which in turn reduces IL-12production and thence downstream IFNγ production from NK cells.

The in vivo experiments for the α-GalCer analogues show that thethioamide and carbamate derivatives, 6 and 8, respectively, both displaya cytokine bias towards IFNγ compared with α-GalCer, resulting from areduction in IL-4 production relative to the parent α-GalCer 1, ratherthan an increase in IFNγ production, which in both cases was similar tothat generated by α-GalCer 1. Results for the ThrCer derivatives weremore significant in that these displayed a similar but more pronouncedtrend. Compared with α-GalCer 1, ThrCer 2 is a weaker activator of iNKTcells, although it displays a similar cytokine profile. ThrCer-thioamide9 and ThrCer-carbamate 11 displayed no IL-4 production, when assayed at2 h; however they showed levels of IFNγ production at 18 h, which werehigher than those shown for ThrCer 2 and only four times lower than thatdisplayed by α-GalCer 1.

Both the α-GalCer and ThrCer analogues induced DC maturation to asimilar degree as the parent α-GalCer and ThrCer compounds in wildtypemice but not in CD1d−/− mice. In terms of binding affinity of the TCRfor glycolipid-loaded hCD1d, K_(d) values for the hCD1d-carbamate-TCRcomplexes in both series were comparable to those measured for theparent compounds. The iNKT cell TCR exhibited slightly lower bindingaffinity for hCD1d loaded with α-GalCer-thioamide 6 compared withhCD1d/α-GalCer, whereas binding affinity for hCD1d loaded withThrCer-thioamide 9 was interestingly an order of magnitude lower thanthat for hCD1d/ThrCer 2. The binding kinetics experiment showed thatthese lower binding affinities are a consequence of a faster off rateand a slower on rate of the hCD1d-thioamide-TCR complex compared to CD1dcomplexes with the parent molecules. This observation is comparable tothe kinetics displayed by the Th2-biasing α-GalCer analogue, OCH9 whoseweak binding (K_(d) of 123±9.08 μM) was attributed to both, a decreasein the k₀ (2.3×10⁴±1×10³ M⁻¹ s⁻¹), as well as an increased k_(off)(2.67±0.12 s⁻¹). These iNKT cell TCR binding affinity data for theCD1d-glycolipid complexes do not show a clear correlation with theobserved cytokine response.

A recent study made a direct comparison between the Th2-biasing OCH9glycolipid 12 and the Th1-biasing C-glycosyl analogue of α-GalCer 13. Itshowed that both OCH9 12 and the C-glycosyl analogue of α-GalCer 13displayed weaker interactions than α-GalCer with the iNKT cell TCRs. Thedifferences in cytokine response profiles was attributed to otherfactors including their differing pharmacokinetics properties, and theirability to transactivate other cytokinereleasing T-lymphocytes such asNK cells. When such transactivation requires prolonged lifetime afterinitial injection, metabolically more stable glycolipid analogues shouldfunction better. A similar observation was made with the neoglycolipidα-carba-GalCer, which induces a Th1-biased cytokine response profileupon iNKT cell activation. Having replaced the amide residue withmetabolically more stable functionality, all three structural analogues,and in particular the non-glycosidic threitol derivatives 9, 10 and 11,which are also not susceptible to glycosidase-mediated hydrolysis, wereexpected to exhibit prolonged lifetimes in vivo, and for this reason, itwas postulated that this might lead to increased IFNγ production. Thishypothesis appears to have been borne out, at least in part, in theThrCer series with the thioamide and carbamate analogues. In bothα-GalCer and ThrCer series, the urea analogues displayed poor activityand binding and kinetics data for these two substrates wereunattainable, which may suggest that the additional NH functionalityincorporated into the acyl chain disrupts glycolipid binding andsubsequent presentation.

Ever since it was demonstrated that α-GalCer 1 functions as a potentCD1d agonist, numerous structural modifications have probedstructure-activity relationships and led to the discovery of CD1dagonists that are capable of polarizing cytokine production. Structuralvariation around the amide bond in 1 has to-date received scantattention. To this end, thioamide, carbamate and urea analogues ofα-GalCer and its non-glycosidic analogue, ThrCer, were prepared and aninvestigation of their biological activity was conducted. Whilst thecarbamate and thioamide analogues of α-GalCer are similar in behaviourto the parent molecule, the same changes in ThrCer, led to twosubstrates that display a markedly different cytokine response profileupon iNKT cell activation. This study shows for the first time thatamide isosteres of CD1d agonists can be used to elicit significantchanges in cytokine response. Although the factors that govern thecytokine profile are likely multifactorial, it is postulated thatproviding the glycolipid binds in a viable conformation for presentationto iNKT cell TCRs, increased metabolic stability is important forprolonged activation of iNKT cells. By this reasoning, the thioamideanalogue of the C-glycosyl α-GalCer derivative are worthy ofinvestigation.

Biology—Cyclic Analogues

Binding affinity of iNKT TCR to various compounds, as noted below, wasassessed.

C1R-hCD1d cells were pulsed with each ligand noted above and theaffinity of iNKT TCR-tetramer was determined by flow cytometry asmeasured by median Fluorescent Intensity (MFI). The results are shown inFIG. 6. The supernatant was tested for IFNγ by ELISA, and the resultsare shown in FIG. 7. Mice (n=3) were immunized with 0.1 μg of eachcompound on day −28. Splenocytes were cultured for 60 hours in thepresence of α-GalCer and the supernatants were tested for IFNγ by ELISA,the results are shown in FIG. 8.

Following immunization with 800 μg OVA and 1 μg lipids i.v. at day −7,splenocytes were cultured in vitro in the presence of OVA specific MHC I(A) and MHCII (B) peptides. IFNγ release was determined at 18 hours byELISpot and expressed as the number of spot per million splenocytes. Theresults are shown in FIGS. 9A and 9B, respectively.

Adjuvant activity of ThrCer 6 and ThrCer 7 were assessed. Followingimmunization with 800 μg OVA and 1 μg lipids i.v. at day −7, mice werebled and sera tested by ELISA for the presence of OVA-specific IgGs. Theresults are shown in FIG. 10.

Biology Studies of IMM47, IMM60, IMM70, and IMM80

Mice and reagents: C57BL/6 wild-type and CD1d^(−/−) (NKT deficient) micewere used. Animal experiments were carried out under the authority of aU.K. Home Office Project License. Lipid compounds α-GalCer, IMM47(threitolceramide), IMM60 (ThrCer 6-membered ring), IMM70 (ThrCer7-membered ring), and IMM80 (ThrCer 8-membered ring), were solubilizedin vehicle (150 mM NaCl and 0.5% Tween 20; Sigma, UK). Hiltonol (PolyI:C; [Oncovir Inc, USA]) was diluted in phosphate buffered saline (PBS;Oxoid, UK) prior to injection.

In vitro and in vivo activation of iNKT cells: For in vitrore-stimulation of iNKT cells, 5×10⁵ C57BL/6 splenocytes were loaded witheither 100 ng/ml, 10 ng/ml or 1 ng/ml α-GalCer or vehicle for 48 hoursand the presence of IFNγ in supernatant determined by ELISA(eBioscience).

For in vivo activation of iNKT cells, C57BL/6 wild-type or CD1d^(−/−)(NKT cell deficient) mice were injected intravenously with 1 μg lipidsand blood sera taken at 2 hours or 18 hours and the presence of IL-4 andIFNγ determined by ELISA (eBioscience).

Protein expression and purification: hCD1d and β2m were refolded withα-GalCer, IMM47, IMM60, IMM70 or IMM80 by oxidative chromatography. Insummary, CD1d and β2m were overexpressed in E. coli BL21 using aprokaryotic expression system (Novagen, UK). The individual proteinswere purified from inclusion bodies, then refolded with thecorresponding lipid. The complex was biotinylated and purified.

Preparation of human iNKT TCR: Soluble TCR was prepared according to theprotocol described by McCarthy et al., J Exp Med, 204 (5), 1131-44(2007), where both Vα24 and Vβ11 chains were individually overexpressedin E. coli and purified from the inclusion bodies, then refolded andpurified to generate the TCR heterodimers.

Surface plasmon resonance: SPR experiments were performed with a model3000 BIAcore to measure the affinity and kinetics of iNKT TCR binding tohCD1d-ligand complexes. In brief, approximately 1000RU of thebiotinylated hCD1d-lipid complexes, were immobilized ontostreptavidin-coated CMS sensor chips (BIAcore). Injections of purifiedTCR serial dilutions were passed on the immobilized hCD1d-lipid at aflow rate of 10 μl/min for the equilibrium binding experiments, orSOW/min for the kinetics experiments. The K_(d) values were calculatedby fitting the data from the equilibrium binding experiment to anon-linear regression saturation binding model (GraphPad Prism 5.0),whereas the k_(off) data were estimated from the kinetics experiments byfitting the data to the 1:1 Langmuir binding model using the BlAevaal3.1 software (BIAcore).

Comparative Analysis of Alpha-GalCer, ThrCer, IMM60, and IMM70 on TumorRegression

Mice were injected with 1×10⁶ EG7 cells (EL4 cells transfected with fulllength ovalbumin). Four days later (when tumor was palpable) the micewere immunized with 800 μg of ovalbumin (OVA) alone or various lipids:(alpha-GalCer, ThrCer, IMM60, and IMM70. Mean tumor size was assessed atvarious days after challenge. As summarized in FIG. 19, IMM60 was theonly compound of those tested that induced regression of the establishedtumour.

In Vivo Anergy of iNKT Cells

The following experiment was designed to assess the in vivo anergy ofiNKT cells as defined by their ability to enhance antigen-specific T-and B-cell responses. The experimental design is set forth in FIG. 12.Mice were pre-conditioned at day −28 with individual lipids shown inFIG. 12 (100 ng i.v. of either alphaGalCer, IMM47, IMM60, or IMM70).Eighteen hours later, mice were bled to assess iNKT cell activation, asdefined by IFN-gamma (determined by ELISA(eBioscience)), results shownin FIG. 13. At day zero (28 days after lipid preconditioning),expression of the iNKT cell activation marker PD1 was measured insplenic iNKT cells (FIG. 14). These two results show that IMM60 inducesa stronger iNKT cell activation than IMM47, IMM70, or even alphaGalCer.(This observation is consistent with IMM60's ability to induce strongerT and B cell responses after 7 day priming as described below.) However,IMM60 also caused enhanced DC lysis and enhanced anergy as describedbelow.

As indicated in FIG. 12, immunization was performed at day zero with 800μg OVA and alphaGalCer (for all mice). Eighteen hours later, iNKT cellactivation was analyzed by IFNγ expression by ELISA (FIG. 15). At day 7,T cell responses were analyzed (FIG. 16).

The results in FIG. 15 confirmed previous findings that IMM47 caused theweakest iNKT cell anergy (as defined by the highest amount of IFNγ).AlphaGalCer and IMM60 induced the highest iNKT cell anergy, while IMM70pre-conditioned mice had functional iNKT cells (as shown by theintermediate amount of IFNγ). The results shown in FIG. 16 indicate thatIMM70 preconditioned mice had the highest T-cell response.

Lysis of Dendritic Cells Pulsed with IMM47, IMM60 and IMM70

The following experiments were performed to analyze the extent to whichIMM47, IMM60 and IMM70 cause lysis of human dendritic cells.

Monocyte-derived dendritic cells and human iNKT cells were cultured withvarious concentrations of each lipid for 40 hours and then PI stained todetermine percent dendritic cell lysis (and percent survival). Theresults shown in FIG. 17 show that IMM47 caused the lowest dendriticcell killing (by observing the percent live dendritic cells in thesample) and that IMM60 caused the highest dendritic cell killing.Interestingly, at concentrations lower than 5 ng/ml, the percentage ofDC lysis obtained with IMM70 is very similar to the results obtainedwith IMM47, even though IMM70 is the stronger agonist.

Direct T- and B-Cell Priming by IMM47, IMM60 and IMM70

Experiments were performed to analyze the ability of IMM47, IMM60 andIMM70 to induce T- and B-cell responses. The experimental design is setforth in FIG. 18. Briefly, ovalbumin plus 1 microgram of one of thelipids were used to immunize mice at day zero, and blood/cells weredrawn and analyzed at the indicated times. The data provided in FIG. 11show that IMM60 induced stronger T- and B-cell responses compared toIMM47 and IMM70, as measured by CD8+Kb tetramer+ cells and serum OVA IgGlevels.

1. A compound having a structure (I):

wherein R¹ is C₅-C₂₅ alkyl, C₅-C₂₅ alkenyl, C₅-C₂₅ alkynyl, C₅-C₂₅heteroalkyl, C₅-C₂₅ heteroalkenyl, or C₅-C₂₅ heteroalkynyl; R² and R³are each independently selected from H, OH, SH, amino or substitutedamino; R⁴ is C₅-C₂₀ alkyl, C₅-C₂₀ alkenyl, C₅-C₂₀ alkynyl, C₅-C₂₀heteroalkyl, C₅-C₂₀ heteroalkenyl, or C₅-C₂₀ heteroalkynyl; R⁶ and R⁵are each independently selected from H, alkyl, and alkenyl, or R⁶ and R⁵together form a 6-, 7-, or 8-membered cycloalkyl or cycloalkenyl ring; Xis O, S, SO₂, SO(NH), SO(N(alkyl)), NH, N(alkyl), or CH₂; Y is O, NH,N(alkyl), or S; Z is O, S, NH, N(alkyl), or CH₂; with the proviso thatwhen Y and X are each O and R⁵ and R⁶ are each H, Z is not CH₂; and whenY is O, R⁶ and R⁵ together form a 6-membered cycloalkyl ring, and Z isCH₂, the cycloalkyl ring is not substituted with —CH₂OH, —OH, —CH₃, or—CH₂OCH₃, or a salt, ester, solvate, or hydrate thereof.
 2. Thecompound, salt, ester, solvate, or hydrate of paragraph 1 having astructure (IA), (TB), (IC), or (ID):

wherein n is 1, 2, or 3; m is 0, 1, or 2; p is 1 or 2; and the dashedline is an optional double bond.
 3. The compound, salt, ester, solvate,or hydrate of paragraph 1, wherein R⁶ is H.
 4. The compound, salt,ester, solvate, or hydrate of paragraph 1, wherein R⁶ is alkyl.
 5. Thecompound, salt, ester, solvate, or hydrate of paragraph 1, wherein R⁶ isalkenyl.
 6. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 and 3 to 5, wherein R⁵ is H.
 7. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 and 3 to 5, wherein R⁵ isalkyl.
 8. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 and 3 to 5, wherein R⁵ is alkenyl.
 9. The compound, salt,ester, solvate, or hydrate of any one of paragraphs 1 to 8, wherein Z isCH₂.
 10. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 8, wherein Z is O.
 11. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 8, wherein Z is S. 12.The compound, salt, ester, solvate, or hydrate of any one of paragraphs1 to 8, wherein Z is NH.
 13. The compound, salt, ester, solvate, orhydrate of any one of paragraphs 1 to 8, wherein Z is N(alkyl).
 14. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to8, wherein Z is CH₂.
 15. The compound, salt, ester, solvate, or hydrateof any one of paragraphs 1 to 14, wherein X is O.
 16. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 14,wherein X is S.
 17. The compound, salt, ester, solvate, or hydrate ofany one of paragraphs 1 to 14, wherein X is SO₂.
 18. The compound, salt,ester, solvate, or hydrate of any one of paragraphs 1 to 14, wherein Xis SO(NH).
 19. The compound, salt, ester, solvate, or hydrate of any oneof paragraphs 1 to 14, wherein X is SO(N(alkyl)).
 20. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 14,wherein X is NH.
 21. The compound, salt, ester, solvate, or hydrate ofany one of paragraphs 1 to 14, wherein X is N(alkyl).
 22. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 14,wherein X is CH₂.
 23. The compound, salt, ester, solvate, or hydrate ofany one of paragraphs 1 to 22, wherein Y is O.
 24. The compound, salt,ester, solvate, or hydrate of any one of paragraphs 1 to 22, wherein Yis NH.
 25. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 22, wherein Y is N(alkyl).
 26. The compound, salt,ester, solvate, or hydrate of any one of paragraphs 1 to 22, wherein Yis S.
 27. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 26, wherein R² is H.
 28. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 26, wherein R² is SH.29. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 26, wherein R² is amino or substituted amino.
 30. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to26, wherein R² is OH.
 31. The compound, salt, ester, solvate, or hydrateof any one of paragraphs 1 to 30, wherein R³ is H.
 32. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 30,wherein R³ is SH.
 33. The compound, salt, ester, solvate, or hydrate ofany one of paragraphs 1 to 30, wherein R³ is amino or substituted amino.34. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 30, wherein R³ is OH.
 35. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 34, wherein at leastone of R² and R³ is OH.
 36. The compound, salt, ester, solvate, orhydrate of paragraph 35, wherein R² and R³ are each OH.
 37. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to36, wherein R¹ is C₁₅-C₂₅ alkyl.
 38. The compound, salt, ester, solvate,or hydrate of paragraph 37, wherein R¹ is C₂₃alkyl or C₂₄alkyl.
 39. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to36, wherein R¹ is C₁₅-C₂₅ heteroalkyl.
 40. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 36, wherein R¹ isC₁₅-C₂₅ alkenyl.
 41. The compound, salt, ester, solvate, or hydrate ofparagraph 40, wherein R¹ is C₂₃alkenyl or C₂₄alkenyl.
 42. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 36,wherein R¹ is C₁₅-C₂₅ heteroalkenyl.
 43. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 36, wherein R¹ isC₁₅-C₂₅ alkynyl.
 44. The compound, salt, ester, solvate, or hydrate ofparagraph 43, wherein R¹ is C₂₃alkynyl or C₂₄alkynyl.
 45. The compound,salt, ester, solvate, or hydrate of any one of paragraphs 1 to 36,wherein R¹ is C₁₅-C₂₅ heteroalkynyl.
 46. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 45, wherein R⁴ isC₁₀-C₂₀ alkyl.
 47. The compound, salt, ester, solvate, or hydrate ofparagraph 46, wherein R⁴ is C₁₃alkyl.
 48. The compound, salt, ester,solvate, or hydrate of any one of paragraphs 1 to 45, wherein R⁴ isC₁₀-C₂₀ heteroalkyl.
 49. The compound, salt, ester, solvate, or hydrateof any one of paragraphs 1 to 45, wherein R⁴ is C₁₀-C₂₀ alkenyl.
 50. Thecompound, salt, ester, solvate, or hydrate of paragraph 49, wherein R⁴is C₁₃alkenyl.
 51. The compound, salt, ester, solvate, or hydrate of anyone of paragraphs 1 to 45, wherein R⁴ is C₁₀-C₂₀ heteroalkenyl.
 52. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to45, wherein R⁴ is C₁₀-C₂₀ alkynyl.
 53. The compound, salt, ester,solvate, or hydrate of paragraph 52, wherein R⁴ is C₁₃ alkynyl.
 54. Thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to45, wherein R⁴ is C₁₀-C₂₀ heteroalkynyl.
 55. The compound, salt, ester,solvate, or hydrate of paragraph 1 having a structure selected from thegroup consisting of:


56. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 55, having a structure of (IE):


57. The compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 56 that is purified and isolated.
 58. A compositioncomprising the compound, salt, ester, solvate, or hydrate of any one ofparagraphs 1 to 57 and a pharmaceutically acceptable excipient.
 59. Amethod of activating an NKT cell comprising contacting the cell with thecompound, salt, ester, solvate, or hydrate of any one of paragraphs 1 to57 or the composition of paragraph 58 in an amount effective to activatethe NKT cell.
 60. The method of paragraph 59, wherein activating the NKTcell comprises one or more of inducing secretion of a cytokine from theNKT cell, stimulating proliferation of the NKT cell, and upregulatingexpression of a cell surface marker on the NKT cell.
 61. The method ofparagraph 60, wherein the activating comprises inducing secretion of acytokine and the cytokine is one or more of IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, IL-13, IL-15, TNF-α, TNF-β, and IFN-γ.
 62. The method ofparagraph 60, wherein the activating comprises upregulating at least onecell surface marker selected from CD69, CD25, an IL-12 receptor andCD40L.
 63. The method of any one of paragraph 59 to 62, wherein thecontacting comprises administering the compound, salt, ester, solvate,or hydrate of any one of paragraphs 1 to 57 to a subject in need of NKTcell activation.
 64. The method of paragraph 63, wherein the subjectsuffers from cancer.
 65. The method of paragraph 64, wherein the subjectsuffer from a cancer selected from basal cell carcinoma, breast cancer,leukemia, Burkitt's Lymphoma, colon cancer, esophageal cancer, bladdercancer, gastric cancer, head and neck cancer, hepatocellular cancer,Hodgkin's Lymphoma, hairy cell leukemia, Wilms' Tumor, thyroid cancer,thymoma, thymic carcinoma, testicular cancer, T-cell lymphoma, prostatecancer, non-small cell lung cancer, liver cancer, renal cell cancer, andmelanoma.
 66. The method of any one of paragraphs 63 to 65, furthercomprising administering a second therapeutic to the subject.
 67. Themethod of paragraph 66, wherein the second therapeutic is achemotherapeutic or an immunotherapeutic agent.
 68. The method ofparagraph 67, wherein the immunotherapeutic is a cancer vaccine.
 69. Themethod of paragraph 67, wherein the immunotherapeutic is a cancerantigen.
 70. The method of paragraph 69, wherein the cancer antigen isselected from MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13,GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1,RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A,MAGE-C₁, MAGE-C₂, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1,SSX-4, SSX-5, SCP-1, CT-7, alpha-actinin-4, Bcr-Abl fusion protein,Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusionprotein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusionprotein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3,neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras,N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, Lage-1, Mage-C₂,NA-88, /Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1,TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom/Mel-40,p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR,Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigensE6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3,c-met, nm-23H₁, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,.beta.-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase,43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18,NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.71. The method of paragraph 67, wherein the immunotherapeutic is apolynucleotide encoding a cancer antigen.
 72. The method of paragraph71, wherein the polynucleotide is in a vector.
 73. The method ofparagraph 66, wherein the second therapeutic is selected from aspirin,sulindac, curcumin, alkylating agents including: nitrogen mustards, suchas mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; epipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platinumcoordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; non-steroidal antiandrogens such as flutamide; kinaseinhibitors, histone deacetylase inhibitors, methylation inhibitors,proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants,telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, statinhibitors, herceptin, alemtuzumab, gemtuzumab, rituximab, ibritumomabtiuxetan, imatinib, erlotinib, cyclophosphamide, infliximab,adalimmumab, basiliximab, anti CD40/CD40L antibody, anti-CTLA-4 blockingantibody, soluble LAGS based immune modulator, MPL, CpG, single-strandedRNA, nucleotides, nucleotide analogue, CL087, loxoribine,polyinosine-polycytidylic acid, flagellin, resiquimod, immiquimod,gardiquimod, NOD ligands, muramyl dipeptide, murabutide, peptidoglycan,muramyldipeptide, oseltamivir phosphate, Amphotericin B, andpalivizumab.
 74. The method of any one of paragraphs 66 to 73, whereinthe second therapeutic and the compound, salt, ester, solvate, orhydrate is administered simultaneously.
 75. The method of paragraph 74,wherein the second therapeutic and the compound, salt, ester, solvate,or hydrate are co-formulated.
 76. The method of any one of paragraphs 66to 73, wherein the second therapeutic and the compound, salt, ester,solvate, or hydrate are administered sequentially.
 77. The method ofparagraph 76, wherein the second therapeutic is administered before thecompound, salt, ester, solvate, or hydrate.
 78. The method of paragraph76, wherein the second therapeutic is administered after the compound,salt, ester, solvate, or hydrate.
 79. A method of treating a subjectsuffering from cancer comprising administering to the subject acompound, salt, ester, solvate or hydrate of any one of paragraphs 1 to57 in an amount effective to treat the cancer.
 80. The method ofparagraph 79, wherein the subject is human.
 81. The method of paragraph79 or 80, wherein the cancer is selected from basal cell carcinoma,breast cancer, leukemia, Burkitt's Lymphoma, colon cancer, esophagealcancer, bladder cancer, gastric cancer, head and neck cancer,hepatocellular cancer, Hodgkin's Lymphoma, hairy cell leukemia, Wilms'Tumor, thyroid cancer, thymoma, thymic carcinoma, testicular cancer,T-cell lymphoma, prostate cancer, non-small cell lung cancer, livercancer, renal cell cancer, and melanoma.
 82. The compound, salt, ester,solvate or hydrate of any one of paragraphs 1 to 57 for treating cancer.83. Use of the compound, salt, ester, solvate or hydrate of any one ofparagraphs 1 to 57 in the manufacture of a medicament for treatingcancer.